patent text
5,496,720
[7m <=2> [0m GET 1st DRAWING SHEET OF 4
Mar. 5, 1996
Parthenogenic oocyte activation
INVENTOR: Susko-Parrish, Joan L., 207 Stone Ter., Monona, Wisconsin 53716
Northey, David L., 6706 Clovernook Cir., Middleton, Wisconsin 53562
Leibfried-Rutledge, M. Lorraine, 321 Glen Thistle Ct., Madison, Wisconsin
53705 Stice, Steven L., 204 Mohican Pass, DeForest, Wisconsin 53532
ASSIGNEE-AFTER-ISSUE: Date Transaction Recorded: Feb. 03, 1997
ASSIGNMENT OF ASSIGNOR'S INTEREST (SEE DOCUMENT FOR DETAILS).
ABS GLOBAL, INC. 6908 RIVER ROAD DEFOREST, WISCONSIN 53532
Reel & Frame Number: 8328/0363
APPL-N0: 16,703
FILED: Feb. 10, 1993[H
US-CL: 435#240.2; 424#93.2; 600#33
CL: 435;424;600
SEARCH-FLD: 435#240.2, 240.25, 240.3, 240.31, 172.1; 935#89; 424#94.6,
682, 722,93R; 514#261; 600#33
REF-CITED:
U.S. PATENT DOCUMENTS
[7m <=3> [0m 4,994,384 2/1991 * Prather et al. 435#172.2
[7m <=4> [0m 5,096,822 3/1992 * Rosenkrans 435#240.1
OTHER PUBLICATIONS
Wickramasinghe et al, Dev. Biol. 152(1). pp. 62-74 (1992): Biosis abstract
#94090457.
DeSutter et al, J. Assist Reprod. Genet, 9(4) pp. 328-337 (1992): Biosis
Abstract #95036402.[H
(1991). Collas, P. and J. M. Robl. 1990. "Factors affecting the
efficiency of nuclear transplantation in the rabbit embryo." Biol. Reprod.
43:877-884. Cuthbertson, K. S. R. 1983. "Parthenogenic activation of mouse
oocytes in vitro with ethanol and benzyl alcohol." J. Exp. Zool.
226:311-314. First, N. L., Leibfried-Rutledge, M. L., Northey, D. L. and
Nuttleman, P. R. 1992. "Use of in vitro matured oocytes 24 h of age in
bovine nuclear transfer." Theriogenology 37:211. First, N. L. and R.
Prather. 1991. "Genomic Potential in Mammals," Differentiation, 48:1-8.
Fissore, R. A. and J. M. Robl. 1992. "Intracellular Ca2 + response of
rabbit oocytes to electrical stimulation." Mol. Reprod. Dev. 32:9-16.
Graham, C. F. 1969. "The fusion of cells with one and two cell mouse
embryos," Wistar Inot. Symp. Monogr., 9:19. Graham, C. F. 1970.
"Parthenogenic mouse blastocysts." Nature 242:475-476. Graham, C. F. 1974.
"The production of parthenogenic mammalian embryos and theiruse in
biological research." Biol. Rev. 49:399-422. Gray, K. R., K. R. Bondioli
and C. L. Betts. 1991. "The commercial application of embryo splitting in
beef cattle." Theriogenology 35:37-45. Kaufman, M. H. 1981.
"Parthenogenesis: a system facilitating understanding of factors that
influence early mammalian development." Prog. in Anat., vol. 1, 1-34.[H
he role of calcium in exocytosis and cell cycle activation in the mouse
egg." Dev. Biol. 149:80-89. Kubiak, J. 1989. "Mouse Oocytes Gradually
Develop the Capacity for Activation during the Metaphase II Arrest." Dev.
Biol. 136:537-545. Masui, Y. and Markert, C. L. 1971. "Cytoplasmic control
of nuclear behavior during meiotic maturation of frog oocytes." J. Exp.
Zool. 177, 129-146. McGrath, J. and Solter, D. 1983. "Nuclear
Transplantation in the Mouse Embryo byMicrosurgery and Cell Fusion."
Science, vol. 220, 1301-1302. Nagai, T. 1987. "Parthenogenic activation of
cattle follicular oocytes in vitro with ethanol." Gamete Res. 16:243-249.
Nurse, P. 1990. "Universal control mechanism regulating onset of M-phase."
Nature 344:503-508. Onodera, M. and Y. Tsunoda. 1989. "Parthenogenetic
activation of mouse and rabbit eggs by electric stimulation in vitro."
Gamete Research 22:277-283. Ozil, J. P. 1990. "The parthenogenetic
development of rabbit oocytes after repetitive pulsatile electrical
stimulation." Development 109:117-127. Rickords, L. F. and White, K. L.
1992. "Electrofusion-induced intracellular Ca +2 flux and its effect on
murine oocyte activation." Mol. Reprod. Dev. 31:152-159. Siracusa, G.,
Whittingham, D. G., Molinaro, M. and Vivarelli, E. 1978. "Parthenogenic
activation of mouse oocytes induced by inhibitors of protein[H
synthesis." J. Embryol. exp. Morph. 43:157-166. Sirard, M. A., J. J.
Parrish, C. B. Ware, M. L. Leibfried-Rutledge and N. L. First. 1988. "The
culture of bovine oocytes to obtain developmentally competent embryos."
Biol. Reprod. 39:546-552. Steinhardt, R. A., Epel, D. and Yanagimachi, R.
1974. "Is calcium ionophore a universal activator for unfertilized eggs?"
Nature 252:41-43. [7m Stice, [0m S. L. and [7m Robl, [0m J. M. 1990.
"Activation of mammalian oocytes by a factor obtained from rabbit sperm."
Mol. Reprod. Dev. 25:272-280. Stice, S. L. and Keefer, C. 1992. "Improved
developmental rates for bovine nucleus transfer embryos using cold shock
activated oocytes." Biol. Reprod. 42 (Suppl 1):166. Surani, M. A. H. and
Kaufmann, N. H. 1977. "Influence of extracellular Ca2 + andMg2 + ions on
the second meiotic division of mouse oocytes: Relevance to obtaining
haploid and diploid parthenogenetic embryos." Dev. Biol. 59:86-90. Swann,
K. 1990. "A cytosolic sperm factor stimulates repetitive calcium increases
and mimics fertilization in hamster eggs." Development 110:1295-1302.
Tarkowski, A. K., Witkowska, A. and Nowicka, J. 1970. "Experimental
parthenogenesis in the mouse." Nature 226:162-165. Tarkowski, A. K. 1975.
"Recent studies on parthenogenesis in the mouse." In: TheDevelopmental
Biology of Reproduction (C. L. Markert, E. J. Papaconstantinon, eds.), pp.
107-129, New York: Academic Press. Ware, C. B., Barnes, F. L.,
Maiki-Laurila, M. and First, N. L. 1989. "Age[H ependence of bovine
oocyte activation." Gamete Res. 22:265-275. Whitaker, M. and Irvine, F. R.
1984. "Inositol 1,4,5-trisphophate microinjectionactivates sea urchin
eggs." Nature (London) 312:636-639. Yang, S., Jiang, S. and Shi, Z. 1992.
"Improved activation by combined cycloheximide and electric pulse
treatment of bovine follicular oocytes matured in vitro for 23-24 hours."
Biol. Reprod. 42(Suppl 1): 117. Yang, X. et al., 1990, "Potential of
hypertonic medium treatment for embryo micromanipulation: II. Assessment
of nuclear transplantation methodology, isolation, subzona insertion and
electrofusion of blastomeres," Mol. Reprod. Dev. 27:118-129. Yang, X. et
al., 1991, "Nuclear transfer in rabbits and cattle by electric
pulse-induced fusion of blastomeres to enucleated oocytes," Theriogenology
35:298 (abs).
PRIM-EXMR: Robinson, Douglas W.
ASST-EXMR: Dadio, Susan M.
LEGAL-REP: Ross & Stevens
ABST:
A process of parthenogenic activation of mammalian oocytes which
includes increasing intercellular levels of divalent cations in the oocyte; and reducing phosphorylation of cellular proteins in the oocyte. One method of accomplishing this is by introducing Ca<2 + > free cation, such as ionomycin, to the oocyte
and then preventing phosphorylation of the cellular proteins within the oocyte
by adding a serine-threonine kinase inhibitor, such as 6-dimethylaminopurine
(DMAP).
NO-OF-CLAIMS: 25
EXMPL-CLAIM: [7m <=5> [0m 1
NO-OF-FIGURES: 6
NO-DRWNG-PP: 4
SUM:
FIELD OF THE INVENTION
The present invention is generally directed to an improved process for
cloning or multiplying mammalian embryonic cells and to an improved
process for transferring the nuclei of donor embryonic cells into
enucleated recipient[H
oocytes.The present invention is specifically directed to a process for
parthenogenically activating mammalian oocytes and to the use of the
oocytes. CITATION OF REFERENCES
A full citation of the references appearing in this disclosure can be
found in the section preceding the claims. DESCRIPTION OF THE PRIOR ART
Advanced genetic improvement and selection techniques continue to be
sought in the field of animal husbandry. With specific reference to dairy
cattle, for example, significant increases in milk production have been
made with the wide scale use of genetically superior sires and artificial
insemination. Dairy cows today produce nearly twice as much milk as they
did 30 years ago. Further genetic improvement can be accomplished by the
multiplication of superior or genetically manipulated animals by cloning
using embryonic cells. For purposes of the present invention, the term
"embryonic cell" refers to embryos and cells cultured from embryos
including embryonic stem cells.
It has now become an accepted practice to transplant embryonic cells in
cattle to aid in the production of genetically superior stock. The cloning
of embryonic cells together with the ability to transplant the cloned
embryonic cells makes it possible to produce multiple genetically
identical animals.[H Embryonic cell cloning is the process of
transferring the nucleus of an embryonic donor cell to an enucleated
recipient ovum or oocyte. The clone then develops into a genetically
identical offspring to the donor embryonic cell.
Nuclear Transfer
The ability to produce multiple copies of genetically identical
individuals from embryonic cells derived from a single embryo provides a
means for embryoniccell selection where the cloned lines descending from
one embryo could be selected by progeny testing for further clonal
multiplication. Nuclear transfer creates the possibility of permitting
rapid changes in animal characteristics such as meat and milk production.
Nuclear transfer is one process for producing multiple copies of an
embryo. Reference is made to First and Prather (1991) and U.S. Pat. No.
[7m <=6> [0m 4,994,384 to Prather et al., which are incorporated herein
by refe rence, for a description of nuclear transfer.
Briefly, nuclear transfer involves the transfer of an embryonic cell or
nucleus from an embryonic cell. Either entity is derived from a
multicellular embryo (usually 20 to 64-cell stage) into an enucleated
oocyte, an oocyte with the nucleus removed or destroyed. The oocyte then
develops into a multi-cellularstage and is used to produce an offspring or
as a donor in serial recloning.[H
Cloningby nuclear transfer has great potential for the multiplication of
genotypes of superior economic value (Gray et al., 1991). Nuclear transfer
to produce identical offspring has many advantages over embryo splitting
or embryonic cell aggregation to produce fetal placental chimeras: 1)
multiple copies of superior, genetically identical animals are possible;
2) embryonic cell sexing and cryopreservation may be applied to the
cloning scheme allowing all clones to be of preselected sex; and 3)
embryonic cells from different genetic strains can be frozen and can be
multiplied after testing.
Oocyte Activation
Cattle ovulate spontaneously approximately every 21 days, about 24-36
hours after a surge of luteinizing hormone (LH). In vivo and in vitro
matured oocytes are activated by entry of sperm into the oocyte.
Activation by sperm can occur in bovine oocytes matured in vitro as early
as 15 hours. However, currently oocytes must be matured for more than
about 28 hours to respond to parthenogenicactivation stimuli. This datum
implies that either the sperm provide a factor necessary for oocyte
activation (Whitaker and Irvine, 1984; [7m Stice and Robl, [0m 1990;
Swann, 1990) or that processes that increase intracellular calcium alone
are not sufficient in the bovine oocyte to overcome the cytostatic
factor(s).
[H
The stage of maturation of the oocyte at enucleation and nuclear
transfer is important (First and Prather, 1991). In general, successful
mammalian embryonic cell cloning practices use the metaphase II stage
oocyte as the recipient oocyte. At this stage, it is believed the oocyte
is sufficiently "activatable" to treat the introduced nucleus as it does a
fertilizing sperm.
Activation of mammalian oocytes involves exit from meiosis and reentry
into the mitotic cell cycle by the secondary oocyte and the formation and
migration of pronuclei within the cell. Viable oocytes prepared for
maturation and subsequent activation are required for nuclear transfer
techniques.
Activation requires cell cycle transitions. The Maturation Promoting
Factor complex becomes essential in the understanding of oocyte senescence
and age dependent responsiveness to activation. MPF activity is partly a
function of calcium (Ca<2 + > ). A major imbalance in the components of
the multi-molecular complex which is required for cell cycle arrest may be
responsible for the increasing sensitivity of oocytes to activation
stimuli during aging.
Parthenogenetic Activation
Parthenogenic activation of oocytes may be used instead of
fertilization by sperm to prepare the oocytes for nuclear transfer.
Parthenogenesis is the[H production" of embryonic cells, with or without
eventual development into an adult, from a female gamete in the absence of
any contribution from a male gamete (Kaufman 1981).
Parthenogenetic activation of mammalian oocytes has been induced in a
number of ways. Using an electrical stimulus to induce activation is of
particular interest because electrofusion is part of the current nuclear
transfer procedure. Tarkowski, et al. (1970) reported successful use of
electric shock toactivate the mouse ova while in the oviduct.
Parthenogenetic activation in vitroby electrical stimulation with
electrofusion apparatus used for embryonic cell-oocyte membrane fusion has
been reported ([7m Stice and Robl, [0m 1990; Collas and [7m Robl, [0m
1990; Onodera and Tsunoda, 1989). In the rabbit, with the combined AC and
DC pulse 80 to 90 percent of freshly ovulated oocytes have been
activated(Yang, et al., 1990, 1991). Ozil (1990) used multiple electrical
pulses to induce adequate activation of rabbit oocytes. Adapting this for
nuclear transfer, Collas and Robl (1990) obtained improved development to
term.
It is believed that the most effective activating stimulus would be one
that mimicked the response of mammalian oocytes to fertilization. One such
response of rabbit oocytes is characterized by repetitive transient
elevations in intracellular Ca<2 + > levels followed by rapid return to
base line (Fissore andRobl, 1992), which may explain the improved
development with activation by multiple electrical pulses.
Parthenogenic activation of metaphase II bovine oocytes has proven to
be moredifficult than mouse oocytes. Mouse oocytes have been activated by
exposure to Ca< + 2> -Mg< + 2 > free medium (Surani and Kaufman, 1977),
medium containing hyaluronidase (Graham, 1970), exposure to ethanol
(Cuthbertson, 1983), Ca< + 2 >ionophores or chelators (Steinhardt et al,,
1974; Kline and Kline, 1992), inhibitors of protein synthesis (Siracusa et
al., 1978) and electrical stimulation (Tarkowski et al., 1970). These
procedures that lead to high rates of parthenogenic activation and
development of mouse oocytes do not activate young bovine oocytes and/or
lead to a much lower development rate. Fertilizationand parthenogenic
activation of mouse oocytes is also dependent on post ovulatory aging
(Siracusa et al., 1978).
Activation of bovine oocytes has been reported by ethanol (Nagai,
1987), electrical stimulation (Ware et al., 1989), exposure to room
temperature (Stice and Keefer, 1992), and a combination of electrical
stimulation and cycloheximide(First et al., 1992; Yang et al., 1992).
While these processes are thought to raise intracellular Ca< + 2 >
(Rickord and White, 1992), they are most successful when the oocytes have
been aged for more than 28 hours of maturation (Ware et al., 1989).
SUMMARY OF THE INVENTION[H for parthenogenically activating mammalian
oocytes comprising increasing intracellular levels of divalent cations in
the oocyte and reducing phosphorylation of cellular proteinsin the oocyte.
Reducing phosphorylation can be achieved by inhibiting phosphorylation or
preventing phosphorylation according to procedures explained in this
disclosure. The present invention is also directed to a
parthenogenically-activated oocyte produced by this process.
The present invention is further directed to a process for
parthenogenically activating a 10-52 hour mammalian oocyte comprising
increasing intracellular levels of divalent cations in the oocyte by
introducing a divalent cation into the oocyte cytoplasm, and reducing
phosphorylation of cellular proteins in the oocyte wherein phosphorylation
of cellular proteins is reduced by adding an effective phosphorylation
inhibiting amount of a serine-threonine kinase inhibitor to the oocyte.
The present invention is also directed to a method for transferring a
nucleusfrom a donor embryonic cell to a parthenogenically-activated
recipient oocyte and culturing the resulting nuclear transferred embryo in
vitro or in vivo comprising collecting the embryonic cell; isolating a
membrane-bound nucleus from the embryonic cell; collecting recipient
oocytes from donor animals or their products in vitro; parthenogenically
activating the recipient oocytes, wherein the oocytes are activated by a
process comprising increasing intracellular levels of divalent cations in
the oocyte and reducing phosphorylation of cellular proteins in the
oocyte; transferring the nucleus to the enucleated recipient
parthenogenically-activated oocyte to form a nuclear transferred oocyte;
and forming a single cell embryo from the nuclear transferred oocyte.
The present invention allows nuclear transfer processes to proceed with
younger oocytes such as a 24-hour oocyte, which may produce healthier
embryonic cells. There is evidence indicating that early oocyte activation
allows for better development of the nuclear transplanted cell. The
24-hour oocyte is the approximate age of an in vivo oocyte during natural
fertilization.
Another advantage to activating younger oocytes is the ability, in the
laboratory, to obtain a faster turn around time. Within the procedure of
the current art, a typical oocyte is a 41-43 hour oocyte. Therefore, the
oocyte usedin nuclear transfer technology is typically 17-19 hours older
than an oocyte used within the process of the present invention which, for
example, allows a 24-hour oocyte to be activated.
The younger oocyte potentially allows for tests to be performed in a
shorter time period. Further, the laboratory is operated more efficiently
with faster turnaround of test results. In industry, the use of a younger
oocyte will allowprogeny to be produced in less time.
Further objects, features and advantages of the present invention will
be apparent from the following detailed description when taken in
conjunction with the accompanying drawings.
DRWDESC:
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. 1a is a photograph illustrating a metaphase II bovine oocyte from the
control treatment in Experiment 1.
FIG. 1b is a photograph illustrating the activation of oocytes by ionomycin
in Experiment 1.
FIG. 1c is a photograph illustrating the activation of oocytes by DMAP
alone in Experiment 1.
[H
FIG. 1d is a photograph illustrating the
activation of oocytes following sequential treatment of ionomycin and DMAP
in Experiment 1.
FIG. 2 is a graph illustrating the kinetics of parthenogenic activation of
the bovine oocyte (matured for 24 hours) by ionomycin and 6-DMAP. Oocytes were
treated and samples mounted and fixed for evaluation after 1, 2, 3, 4, or 5
hours of incubation. The nuclear stage was determined by observation with
Nomarski optics.
FIG. 3 is a graph illustrating the effect of time from ionomycin
treatment to6-DMAP treatment on the parthenogenic activation of 24-hour
bovine oocytes (r = 3). Oocytes were mounted after a total of 5 hours
incubation and assessed for DNA form. Activated oocytes which rated as
having clumped chromatin (CC), 1 pronucleus and 2 polar bodies (1PN2PB), 2
pronuclei and 1 polar body (2PN1PB), or 1 pronucleus and 1 polar body
(1PN1PB).
DETDESC:
DETAILED DESCRIPTION OF THE INVENTION
The overall procedure disclosed herein may be described as cloning or
as multiplication of embryonic cells from an embryo by nuclear transfer
followed by a prolonged maintenance period to increase fusion and
developmental rates of multiple genetically identical embryonic cells, and
ultimately, animals.
In vitro matured bovine oocytes require both an increase in Ca<2 + >
and a reduction in phosphorylation to cause activation and subsequent
entry into the cell cycle. Increases in Ca<2 + > cation alone are
sufficient to cause resumption of mitosis and extrusion of the second
polar body, but not pronuclearformation. Additional treatment to reduce
phosphorylation in cellular proteins suppresses second polar body
extrusion and allows the oocyte to continue as if in mitosis. The oocytes
then resume cell cycles and can initiate limited early pregnancy responses
in utero.
Although it is contemplated that the procedure of the present invention
may be utilized on a variety of mammals, the procedure will be described
with reference to the bovine species. However, the present invention does
not restrict the cloning procedure to bovine embryonic cells.
Oocyte
The term "oocyte," as used here for the recipient oocyte, means an
oocyte which develops from an oogonium and, following meiosis, becomes a
mature ovum. It has been found that not all oocytes are equally optimal
cells for efficient nuclear transplantation in mammals. For purposes of
the present invention, metaphase II stage oocytes, matured either in vivo
or in vitro, have been found to be optimal. Mature metaphase II oocytes
may be collected surgically from either nonsuperovulated or superovulated
cows or heifers 24-48 hours past the onset of estrus or past an injection
of human Chorionic Gonadotrophin (hCG) or similar hormone. Alternatively,
immature oocytes may be recovered by aspiration from ovarian follicles
obtained from slaughtered cows or heifers and then may bematured in vitro
in a maturation medium by appropriate hormonal treatment and culturing. As
stated above, the oocyte is allowed to mature in a known maturation medium
until the oocyte enters the metaphase II stage, generally 16-24 hours post
aspiration. For purposes of the present invention, this period of time is
known as the "maturation period." As used herein for calculation of time
periods, "aspiration" refers to aspiration of the immature oocytes from
ovarian follicles.
Maintenance Media
There are a variety of oocyte culture and maintenance media routinely
used for the collection and maintenance of oocytes, and specifically
bovine oocytes. Examples of known media, which may be used for bovine
oocyte culture and maintenance, include Ham's F-10 + 10% fetal calf serum,
Tissue Culture Medium-199 (TCM-199) + 10% fetal calf serum,[H vate
(TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's
media. One of the most common media used for the collection and freezing
of embryonic cells is TCM-199 and 1 to 20% serum supplement including
fetal calf serum, new born serum or steer serum. A preferred maintenance
medium includes TCM-199 with Earle's salts, 10% fetal calfserum, 0.2 mM
Na-pyruvate and 25 ug/ml gentamicin sulphate.
Another maintenance medium is described in U.S. Pat. No. [7m <=7> [0m
5,096,822 to Rosenkrans et al., which is incorporated herein by reference.
This medium, namedCR1, contains the nutritional substances necessary to
support an oocyte.
CR1 contains hemicalcium L-lactate in amounts ranging from 1.0 mM to 10
mM, preferably 1.0 mM to 5.0 mM. Hemicalcium L-lactate is L-lactate with a
hemicalcium salt incorporated thereon. Hemicalcium L-lacatate is
significant in that a single component satisfies two major requirements in
the culture medium: 1) the calcium requirement necessary for compaction
and cytoskeleton arrangement; and 2) the lactate requirement necessary for
metabolism and electron transport. Hemicalcium L-lactate also serves as
valuable mineral and energy source for the medium necessary for viability
of the oocytes.
Examples of the main components in CR1 medium include hemicalcium
L-lactate, sodium chloride, potassium chloride, sodium bicarbonate and a
minor amount of fatty-acidfree bovine serum albumin. Additionally, a
defined quantity of essential and non-essential amino acids may be added
to the medium. CR1 with amino acids is known by the abbreviation "CR1aa."
=[0m
CR1 medium preferably contains the following components in the
following quantities:
sodium chloride-114.7 mM
potassium chloride-3.1 mM
sodium bicarbonate-26.2 mM
hemicalcium L-lactate-5 mM
fatty-acid free BSA-3 mg/ml
Stripping the Oocytes
Prior to activation, the cumulus cells are stripped from the oocytes.
Cumuluscells are non-reproductive or somatic cells which surround the
oocyte and are believed to provide both protection and nutrients needed to
mature the oocyte.[H
The presence of cumulus cells creates a cloud around the oocytes
making it very difficult if not impossible to observe oocytes during the
maturation period.
Cumulus cells are stripped from an oocyte mechanically by pipetting
cumulus enclosed oocytes (CEOs) through the neck of the micropipette
(180-210 mu m innerdiameter) attached to a syringe. Cumulus cells fall off
and denuded oocytes are removed from the maintenance medium. Cumulus cells
are further disaggregated mechanically by pipetting them through the neck
of the micropipette (20-30 mu m inner diameter).
Other methods of stripping cumulus cells from an oocyte include
removing the cells by vortexing approximately 200 mu l of the TALP medium
with the oocyte forapproximately 3 minutes. Alternatively, the cells may
be mechanically stripped by ultrasound techniques known to the art. The
cells may also be stripped enzymatically by the application of proper
enzymes such as trypsin or collagenase according to methods known to the
art of cell culture.
Washing the Oocytes
The oocytes are then washed according to methods known to the art and
moved to a maintenance medium, examples of which are described above. The
time the oocytes are placed in the maintenance medium is designated "0
hour." Thus, for purposes of this disclosure, the age of the oocyte is the
timed from the placement of the oocyte in the medium. A "24-hour oocyte"
is therefore an oocytewhich has been in the medium for 24 hours.
The oocytes are allowed to mature in the medium for approximately 10-50
hours, preferably about 20-26 hours, and most preferably about 24 hours under
maintenance conditions, e.g., 39o C. with 5% CO2 in air and maximal humidity.
Calcium Introduction
The oocyte is then introduced into a medium that causes the
introduction of free calcium ion into the oocyte cytoplasm. Intracellular
calcium concentration can be increased by any of the processes known in
the art such as use of an ionophore such as ionomycin or A23187, by
electric shock, ethanol treatment, caged chelators which can release
calcium inside the cell in response to specific wavelengths, thapsegardin
or depolarizing oocyte membrane (KCl or NH4Cl). Magnesium or other
divalent cations such as strontium and barium may also be introduced to
the medium and oocyte in lieu of Ca<2 + > . Calcium is located in the cell
membrane, mitochondria, endoplasmic reticula and other partsof the cell as
well as externally to the oocyte before being released by the known
processes and introduced as free Ca<2 + > ion into the oocyte
cytoplasm.[H
Without wishing to be restricted to one source of explanation, it
appears that the initial calcium transient appears to be an upstream event
which activates a cascade of cellular changes necessary for resumption of
meiosis and the cell cycle.
Reducing Phosphorylation in Cellular Proteins
The second phase of the invention contemplates the reduction of
phosphorylation in cellular proteins. Cellular proteins are loosely
defined as the proteins within a cell. A non-limiting list of examples
include Maturation Promoting Factor (MPF), Cytostatic Factor (CSF),
cytoskeletal proteins such as tubulin, and cyclins. There are essentially
two ways to reduce phosphorylation in cellular proteins: (1) inhibiting
phosphorylation, and (2) dephosphorylation.
Inhibition of phosphorylation is related to changes in cell cycle
regulators such as MPF and CSF. MPF controls the release of oocytes from
the prophase block, which promotes nuclear envelope disruption, chromosome
condensation, and spindle formation (Masui & Markert, 1971). MPF is a
complex of cdc2 and cyclin Bwhose activity is regulated by specific
phosphorylation and dephosphorylation (Nurse, 1990). CSF arrests the cell
cycle in metaphase, achieved through MPF activity. The c-mos protein, a
component of CSF, may activate or stabilize MPF by regulating the
stability of cyclin B stability.[H
+ > surge induced by ionophore, electric shock or othermethods is
required since Ca< + > induced transient will continue in the absenceof
the initial stimulus. The oocyte is then introduced to a chemical
mechanism that prevents phosphorylation of serine and threonine amino
acids in other proteins within the oocyte allowing a cascade of events to
occur. The cascade includes cortical and zona pellucida reactions
associated with activation.
The chemical mechanism may include a serine-threonine kinase inhibitor
such as 6-dimethylaminopurine (DMAP), staurosporine, 2-aminopurine, and
sphingosine to prevent phosphorylation of serine and threonine which
induces the cascade.
The serine-threonine kinase inhibitor is thought to inhibit MPF and CSF
activity by inhibiting the specific protein kinases that activate and
deactivatethese complexes. Inhibition of phosphorylation allows the oocyte
to escape from metaphase II and continue as if in mitosis, thereby
parthenogenically activatingthe oocyte.
The serine-threonine kinase inhibitor may also be important in
inhibiting phosphorylations necessary for the spindle apparatus (by
inhibiting c-mos) thus inhibiting expulsion of the second polar body.
Further elucidation of the molecular events of activation are necessary.
The need for an initial calcium transient appears to be universal but the
pathways which regulate the[H quent cascade of events leading to
parthenogenic development may be different in bovine oocytes than any
others thus far studied.
The mechanism may also include a phosphatase which dephosphorylizes the
2 amino acids, thus, preventing a further cascade. Phosphatase 2A and
phosphatase 2B have been implicated in the second messenger systems
important in oocyte activation.
Nuclear Transfer
Parthenogenically-activated oocytes can be used in nuclear transfer
processes. Reference is made to U.S. Pat. No. [7m <=8> [0m 4,994,384 to
Prather et al.(Prather et al.), which is incorporated herein by reference
for a general discussion on nuclear transfer techniques.
Culture of Recipient Oocytes
For the successful commercial use of techniques such as genetic
engineering, nuclear transfer or cloning, the process generally requires
collecting immature (prophase I) oocytes from mammalian ovaries obtained
at a slaughterhouse and maturing the oocytes in a maturation medium prior
to activation until the oocyteenters the metaphase II stage, generally
18-24 hours post-aspiration. For purposes of the present invention, this
period of time is known as the "maturation period." As used herein for
calculation of time periods, "aspiration" refers to aspiration of the
immature oocyte from ovarian follicles.
The stage of maturation of the oocyte at enucleation and nuclear
transfer is important (First and Prather, 1991). In general, successful
mammalian embryo cloning practices use the metaphase II stage oocyte as
the recipient oocyte. At this stage, it is believed the oocyte is
sufficiently "activatable" to treat theintroduced nucleus as it does a
fertilizing sperm. In domestic animals, and especially cattle, the oocyte
activation period is between about 16-52 hours, preferably about 28-42
hours, and most preferably about 24 hours post-aspiration.
Approximately 16-24 hours after the initiation of oocyte maturation,
the oocytes are stripped of cumulus cells according to the processes. The
cumulus cells are a mass of somatic cells which surround the oocyte in
vivo. The cumuluscells provide both protection and the nutrients needed to
mature the oocyte.
Activation
The oocytes are then parthenogenically activated according to the
processes discussed previously. For example, the oocytes can be exposed to
an[H iate quantity, e.g., 5 mu M, ionomycin in TL-HEPES for approximately
4 minutes. The oocytes can then be diluted in TL-HEPES with bovine serum
albumin [BSA]. The oocytes can then be placed into an appropriate
activation medium, e.g., CR1, containing DMAP for approximately 3.5-5
hours. Following incubation in DMAP, the oocytes are diluted in TL-HEPES
(1 mg/ml BSA) and incubated in CR1aa.
Micromanipulation of Oocytes
Micromanipulation of the oocytes is performed in a manner similar to
the methods of McGrath and Solter, 1983, which is incorporated herein for
details ofthe micromanipulation technique. Manipulation is performed in
culture dishes in which microdrops of medium are arranged with each dish
containing approximately 100 mu l drops (TL Hepes with Ca and Mg)
containing the oocytes and 20 mu l drops (TL Hepes with Ca and Mg and
20-50% fetal calf serum) to one side containing the cultured embryonic
cells. The addition of between about 1 and 25%fetal calf serum, or other
sera with activity similar to fetal calf serum, to the medium is
beneficial in reducing the attraction, i.e., the adhesiveness, of the
embryonic cells, thereby preventing cell agglutination and allowing easier
handling during micromanipulation.
[H es a cell holding pipette having an
outerdiameter of approximately 90-180 mu m and an inner diameter of
approximately 25-35 mu m, and a beveled, sharpened enucleation
micropipette having an outer diameter of approximately 10 to 45 mu m,
depending upon the size of the embryonic cell. The
parthenogenically-activated oocyte is positioned on the holding pipette so
that the polar body is towards the transfer tip. The polar body and a
small amount of cytoplasm from the region directly beneath the polar body
is removed.
The cells were then enucleated according to the procedures described in
Prather et al. Preferably, enucleation can be verified by methods known to
the art such as by staining with Hoechst 33342, a DNA stain, removing
excess dye andvisualization with ultra-violet (UV) light (excitation
emission). Reference is made to Experiment 8 (infra.) for a more detailed
description of this and the following procedures. Oocytes with no evidence
of metaphase plate (enucleated oocyte) were selected.
The enucleated oocytes are then parthenogenically activated according
to procedures described above. An example of a preferred activation
procedure is described in Experiment 8, in which the oocytes are exposed
to 5 mu M ionomycin in TL-HEPES for approximately 4 minutes. The
enucleated oocytes are then dilutedin TL-HEPES (30 mg/ml bovine serum
albumin [BSA]), and then diluted in TL-HEPES (1 mg/ml BSA). The oocytes
are then placed in an activation medium CR1 containing 1.9 mM D MAP for
approximately 3.5-5 hours. Following the DMAP step, the enucleated oocytes
are again diluted in TL-HEPES (1 mg/ml BSA) and a blastomere, e.g., a
donated nucleus, was inserted next to the oocyte. A slit is made in the
zona pellucida of the oocyte and the embryonic cell is inserted therein.
The cell is pressed against the cytoplasm where it sticks firmly to
thecytoplasmic membrane. Due to the adhesion of the cells, transfer
pipettes are changed frequently.
Cell Fusion
A variety of fusion techniques may be employed for this invention. For
example, the onset of the electricity by electrofusion can induce the
fusion process. Electrofusion is accomplished by providing a pulse of
electricity that is sufficient to cause a transient breakdown of the
plasma membrane. This breakdown of the plasma membrane is very short and
the membrane reforms very rapidly. If two adjacent membranes are induced
to breakdown and upon reformationthe lipid bilayers intermingle, small
channels will open between the two cells. Due to the thermodynamic
instability of such a small opening, it enlarges until the two cells b
ecome one. Reference is made to Prather et al., which is incorporated
herein by reference, for a further discussion of this process. A variety
of electrofusion media can be used including sucrose, mannitol,[H bitol
and phosphate buffer solution.
Fusion can also be accomplished using Sendai virus as a fusigenic agent
(Graham, 1969).
Polyethylene glycol (PEG) may also be used as a fusigenic agent. Under
prescribed conditions, PEG provides excellent fusion results. In one
protocol, the cells are fused in PEG (molecular weight 1,300-1,600 Sigma),
which is mixed in a solution containing TL Hepes (approximately 1:0.25 mu
g/ml) and polyvinyl alcohol (PVA) (approximately 1 mu g/ml), Ca<2 + > and
Mg<2 + > -free. The media containing the cells is then passed through one
or more dilutions (approximately1:1) of the above-described PEG media. The
cell media is then allowed to rest ina culture media , such as TL Hepes
containing fetal calf serum until the cell membranes return to a normal
appearance. Experimental conditions will vary depending upon the products
used.
The following experiments are illustrative of the present invention and
are not intended to limit the invention in any way. EXPERIMENTS
The following procedures are common to one or more of the
experiments.[H
Oocyte Maturation
The chemicals used in the oocyte maturation process were purchased from
SigmaChemical Co., St. Louis, Mo., unless otherwise indicated.
Preparations and concentrations of bovine serum albumin (BSA) are
indicated for each media.
Bovine oocytes were obtained at an abattoir and transported to the
laboratoryin saline (30o-34o C., transport time 2-6 hr). Oocytes from
small follicles (1-6mm) were aspirated and matured according to the
methods described in Sirard et al., 1988. Briefly, oocytes were diluted
out of the follicular fluid with 3 dilutions of TL HEPES modified by
removing glucose, adding 0.22 mM pyruvate and 1 mg/ml of BSA (Fraction V,
Sigma Chemical CO, St. Louis, Mo.).
The oocytes were placed in 50 mu l drops of TCM-199 (Earle's Salts;
Gibco, Grand Island, N.Y.) supplemented with 10% heat treated fetal calf
serum, 0.22 mMpyruvate, 5 mu g/ml FSH-P (Scherring-Plough Animal Health
Corp., Kenilworth, N.J.) and 1 mu g/ml estradiol. Ten oocytes were
incubated in a 50 mu l drop under paraffin oil for 24 hours at 39o C. in
5% CO2 in air humidified atmosphere.
Activation[H vigorously in 200 mu l of TL-HEPES for 3 min. The denuded
oocytes were diluted in 3 changes of TL-HEPES to separate the oocytes from
the cumulus cells. Where indicated, the oocytes were exposed to 5 mu M
ionomycin (5 mM stock in DMSO; Calbiochem, La Jolla, Calif.) in TL-HEPES
(1 mg/ml fatty acid free BSA) for 4 min. The oocytes were then diluted
into TL-HEPES (30 mg/ml fatty acid BSA) for 5 min followed by dilution
into TL-HEPES (1 mg/ml Fraction V BSA). The oocytes were then moved to 50
mu l drops o f embryonic cell development media (U.S. Pat. No. [7m <=9>
[0m 5,096,822 to Rosenkrans et al., with or without DMAP (1.9 mM) for the
indicated times (39o C., 5% CO2 in air). Embryonic cell development media
contained 114.6 mM NaCl, 3.1 mM KCl, 5 mM hemi-calcium lactate, 0.4 mM Na
pyruvate, 1 mM glutamine, 3 mg/ml fatty acid free BSA, MEM nonessential
amino acids and BME amino acids.
Activation and Development Assessment
Activation was evaluated at times indicated in each experiment. The
oocytes were mounted on slides, fixed with acetic acid:alcohol (1:3) and
observed with Nomarski optics. Special care was taken in determining if
metaphase plates were metaphase II or aberrant metaphase III (Kubiak,
1989). Oocytes that contained a metaphase I or II plate were considered
not activated. Oocytes that contained anaphase, telophase or metaphase II
plates or pronuclei were considered to be[H
activated.
In experiments that determined development potential of the
parthenotes, activated oocytes were cultured for 7-8 days in embryonic
cell development media. On the date after activation, initial cleavage was
determined by visual observation. On day 4 of incubation 5% heat treated
fetal calf serum was added to each drop. For some experiments, blastocysts
were mounted and fixed in aceticacid:alcohol (1:3) and stained with 1%
orcein dissolved in 40% acetic acid to determine cell numbers.
Data Analysis
Activation and development data were analyzed with the Statistical
Analysis System statistical package (General Linear Models (Cary, N.C.).
Means were tested by Duncan's Multiple Range Test. EXPERIMENT 1
Effect of Sequential Exposure of Oocytes to Ionomycin and DMAP
Experiment 1 determined the effect of sequential exposure of oocytes to
ionomycin and DMAP. One level of ionomycin (5 mu M) and DMAP (1.9 mM) was
used throughout all studies. The oocytes were cultured for 5 hours after
ionomycin incubation with or without DMAP. Control oocytes were cultured
in embryonic celldevelopment medium alone. Activation was determined after
5 hours of culture.
Reference is made to Table 1 following, which illustrates the effect of
culture in ionomycin and DMAP for 5 hours on parthenogenic activation of
the bovine oocytes matured for 24 hours, and to FIGS. 1a, 1b, and 1c to
illustrate the results of Experiment 1.
TABLE 1
The eflect of culture in Ionomycin and DMAP for
5 hours on parthenogenic activation of the
bovine oocytes matured for 24 hours.<1>
* * % Pronuclear
Treatment N % Activation formation
Control 83 1.4 +/- 1.4 0
Ionomycin (5 mu M) 80 57.8 +/- 7.8 8.9 +/- 7.3
DMAP (2 mM) 93 7.8 +/- 5.5 7.8 +/- 5.5
Ionomycin + DMAP 91 80.5 +/- 13.1 80.5 +/- 13.1[H
n<1> Letters within a column with different letters were different as
tested with Duncan's Multiple Range Test (p < 0.05). -
A metaphase II bovine oocyte from the control treatment is shown in FIG
1a. Ionomycin alone activated the oocytes to a high level but instead of
forming pronuclei the chromosomes reassembled into a metaphase plate as
illustrated in FIG. 1b. As illustrated in FIG. 1c, DMAP alone did not
activate the oocytes but caused the chromosomes to form a tight clump in
the cytoplasm. The sequential treatment of ionomycin and DMAP activated
the oocytes more than either treatmentalone and pronuclear formation was
significantly higher than for the other treatments. It is interesting to
note that these oocytes contain only 1 polar body, illustrated in FIG. 1d.
EXPERIMENTS 2a AND 2b
Effect of Time of Incubation in DMAP on Activation and Development
Experiments 2a and 2b determined the effect of time of incubation in
DMAP on activation and development. In Experiment 2a, oocytes were
incubated in DMAP for15, 30, 60, 150 and 300 minutes. At the end of the
incubation time indicated, oocytes were rinsed in TL-HEPES (1 mg/ml
Fraction V BSA) and placed into embryonic cell development medium until 5
hours had elapsed. At 5 hours, a[H subsample of oocytes was removed and
mounted to determine activation. The remaining oocytes were returned to
the incubator for 7-8 days. Blastocysts on day 7-8 were rated as good or
poor and cell number was determined.
Experiment 2b was similar in design except that oocytes were incubated
for 2 hours, 3 hours, 4 hours, or 5 hours in DMAP.
The results of Experiment 2a are shown in the following Table 2, which
illustrates the effect of time of incubation in DMAP on activation,
pronuclear formation, initial cleavage and parthenogenic development:
TABLE 2
The effect of time of incubation of DMAP on activation,
pronuclear formation, initial cleavage and parthenogenic
development (r = 4)<1>
Length of * * Pronuc- * * Development
6-DMAP * Activa- lear For- * Cleav- to Blasto-
Incubation * tion mation * age cyst
(min) N ( +/- se) ( +/- se) N ( +/- se) ( +/- se)<2>
[H
0
* (4.6) (1.4) * (3.4)
15 70 52.4 1.4 88 7.2 0
* (11.0) (1.4) * (3.2)
30 80 37.8 2.5 103 4.4 0
* (11.4) (2.5) * (2.1)
60 69 22.6 5.4 111 2.3 0.7
* (4.8) (5.2) * (1.5) (0.7)<3>
150 79 32.9 31.6 105 30.9 9.9
* (6.3) (6.5) * (10.4) (4.3)<4>
300 71 76.6 76.6 100 65.6 21.1
* (9.8) (9.8) * (3.2) (1.5)<5>
n<1> Percents within a column with different superscripts are different (P < 0.05). -
n<2> After day 7-8 of culture. -
n<3> Cell numbers for the blastocysts were good-83 (n = 1). -
n<4> Cell numbers for the blastocysts were good -70.8 +/- 7.7 (n = 4);
poor-25 (n = 1). -[H
+/- 4.7 (n = 6); poor-22+/- 13.0 (n = 10). -
Activation by ionomycin was not different from incubation in DMAP for
15 minutes. However, incubation in DMAP for 30-150 minutes resulted in a
lowered activation. It appears that the DMAP is actively inhibiting
activation. Exposureto DMAP for 150 minutes allows all the oocytes that
are activated to progress tothe pronuclear stage. At 300 minutes, there is
a high level of activation and pronuclear formation. Cleavage and
development are very low until the oocytes have been exposed to DMAP for
150 minutes and the rates are significantly higherat 300 minutes.
The results of Experiment 2b are shown in the following Table 3, which
illustrates the effect of time of incubation in DMAP on activation,
pronuclear formation, initial cleavage and parthenogenic development:
TABLE 3
The effect of time of incubation of DMAP on activation,
pronuclear formation, initial cleavage and parthenogenic
development (r = 3)<1>
[H
ment
DMAP * Activa- lear For- * Cleav- to Blasto-
Incubation * tion mation * age cyst
(h) N ( +/- se) ( +/- se) N ( +/- se) ( +/- se)<2>
0 70 67.1 7.1 84 19.2 0
* (8.4) (3.2) * (3.0)
2 72 39.9 34.9 97 21.6 3.3
* (11.2) (6.3) * (1.2) (3.3)
3 72 75.8 75.8 103 66.5 33.3
* (5.9) (5.9) * (5.2) (5.1)
4 70 75.8 75.8 96 63.5 26.5
* (7.8) (7.8) * (3.8) (5.2)
5 71 86.3 86.3 98 70.4 28.9
* (1.6) (1.6) * (9.1) (3.6)
n<1> Percents within a column with different superscripts are different
(P < 0.05). -
n<2> Day 7-8 of culture. -
[H as significantly lower at 2 hours ofDMAP
incubation than ionomycin alone or 3 hours, 4 hours or 5 hours of DMAP
incubation. Pronuclear formation of oocytes treated with ionomycin alone
was significantly lower than any incubation in DMAP. Incubation in DMAP
for 3 hours,4 hours or 5 hours was not different. Initial cleavage of
ionomycin alone or ionomycin + DMAP 2 hours was not different and was
significantly lower than 3 hours, 4 hours or 5 hours of DMAP incubation.
Blastocyst development was not significantly different at 3 hours, 4 hours
or 5 hours of DMAP incubation.
Reference is also made to Table 4, which illustrates the effect of time of
incubation in DMAP on the cell number in resulting parthenote blastocysts:
TABLE 4
The effect of time of incubation in DMAP on the
cell number in resulting parthenote blastocysts (r = 3).
Length * * * * *
of
DMAP
Incuba-
tion (h) N Good ( +/- se) N Poor ( +/- se)[H
2 3 88.7 (6.3) 0 *
3 19 70.1 (6.6) 7 33.1 (4.9)
4 11 73.6 (7.2) 7 40.1 (11.4)
5 15 83.7 (10.0) 4 44 (9.3)
Cell numbers of the blastocyst generated were not different within quality
classification.
EXPERIMENT 3
Kinetics of Activation When Oocytes Were Exposed to Ionomycin Alone or to
Ionomycin Followed by a 3 Hour Incubation in DMAP
Experiment 3 was designed to determine the kinetics of activation when
oocytes were exposed to ionomycin alone or to ionomycin followed by a 3
hour incubation in DMAP. At the end of the 3 hour incubation, oocytes in
DMAP were diluted in TL-HEPES (1 mg/ml Fraction V BSA) and moved to
embryonic cell development medium until mounting. A sample of oocytes from
both treatments weremounted at 1, 2, 3, 4 and 5 hours after exposure to
ionomycin to determine the activation state.
[H , which is a graph
illustrating the kinetics of parthenogenic activation of the bovine oocyte
(matured for 24 hours) by ionomycin and 6-DMAP. When the oocytes were
treated with ionomycin alone, they had resumed meiosis by 1 hour and there
was evidence of polar body expulsion by 2 hours. By 4-5 hours, the oocytes
were arrested in metaphase III. In contrast, when the oocytes were exposed
to the sequential treatment of ionomycin + DMAP, there was evidence of
pronuclear formation by 2 hours and maximum pronuclear formation by 3
hours. There was no evidence of arrest in metaphase III. These data imply
that DMAP works by inhibiting resumption of meiosis and forces the cells
to enter mitosis without the second reduction division. EXPERIMENT 4
Time at which DMAP is Necessary for Parthenogenic Activation Relative
to the Exposure to Ionomycin
Experiment 4 was designed to determine the time at which DMAP is
necessary for parthenogenic activation relative to the exposure to
ionomycin. The oocytes were exposed to ionomycin and placed in embryonic
cell development medium. At 0,1, 2, 2.5, 3 and 4 hours after ionomycin
exposure, oocytes were placed in embryonic cell development medium + DMAP.
At 5 hours post ionomycin exposure, the oocytes were mounted to determine
their activation state.[H s of Experiment 4 are illustrated in FIG. 3,
which shows the effectof time from ionomycin treatment to 6-DMAP treatment
on the parthenogenic activation of 24-hour bovine oocytes. As the interval
from ionomycin treatment to exposure to DMAP increased to more than 1
hour, the oocytes showed an increase in ability to resume meiosis to form
either a second pronucleus or a single pronucleus and 2 polar bodies.
Experiment 3 showed that at 2 hours, oocytes treated with ionomycin alone
had resumed meiosis and ranged from anaphase to polar body expulsion.
These data imply that DMAP may force the chromatin to be converted to
pronuclei or clumped chromatin. EXPERIMENT 5
Initiating and Sustaining Pregnancy With Parthenote Blastocysts
Experiment 5 was designed to determine if parthenote blastocysts could
initiate and sustain a pregnancy. Blastocysts were generated by treatment
with ionomycin followed by a 3 hour incubation in 6-DMAP. After 7 days in
culture, blastocysts that were rated as good or excellent were
nonsurgically transferred to the uterus of Holstein heifers that were in
heat 6-7 days prior to transfer. Two blastocysts were transferred per
recipient, 1 contralateral and 1 ipsilateral to the ovary that had been
palpated to contain the corpus luteum (CL). Cows were checked for signs of
estrus twice daily and Kamar Registered TM Heatmount Detectors were used
to aid in heat detection (Kamar, Inc.; Steamboat[H
Springs, Colo.). Recipients were palpated every 3-4 days after day 21-23 of the cycle. Ultrasound was used to detect uterine vesicles and to assess CL presence.
The results of Experiment 5 are illustrated in the following Table 5,
which illustrates embryonic cell transfer of blastocysts resulting from
parthenogenic activation of bovine oocytes with ionomycin and DMAP.
TABLE 5
Embryonic cell transfer of blastocysts
resulting from parthenogenic activation of
bovine oocytes with ionomycin and DMAP
Total transfers (n) 22
Extended cycle (%) 7 (32.0%)
intra estrual interval (d)
Normal 20.9 +/- 0.5
Extended 29.8 +/- 2.2
n Cycles > 24 days. -
[H stablish prolonged estrous cycles in
bovine. Relatively fewer transfers resulted in prolonged estrous cycles,
CLs and uterine vesicles (32%), than in the mouse (Graham, 1974;
Tarkowski, 1975 (62%); Siracusa et al., 1978). The extended cycles had a
mean of 29.8 +/- 2.2 days.
EXPERIMENT 6
Use of Parthenogenically Activated Aged Oocytes in a Nuclear Transfer
Procedure
Bovine oocytes were obtained and placed in maturation media for
approximately20 hours. The cumulus cells were then stripped off the
oocytes according to the previously described procedures.
The oocytes were then enucleated according to Prather et al. and placed
in CR1aa maturation medium until 40 hours had elapsed. The oocyte were
then placed in CR1aa maturation medium with 1.9 mM DMAP for 2 hours and
incubated at room temperature activation (RTC) conditions (about 25o C.).
Following incubation, embryonic cells were transferred into the oocytes
and the 2 cells were electrically fused together according to the
procedures described in Prather et al., with the exception of the step of
waiting until 42 hours to activate via RTC (25o C.) and adding DMAP prior
to the time of electrofusion. The fused nuclear transfer embryos were then
cultured in maturation medium CR1aa for 6 to 7 days to determine
developmental rates to the blastocyst stage.
Reference is made to table 6 for results of this experiment:
TABLE 6
Development of 42-hour oocyte derived nuclear
transfer (NT) embryos
# NTs to Number Number Usable
Treatment Culture Blastocyst Blastocyst
RTC 146 29 (20%) 11 (8%)
(2 hours)
DMAP & RTC 140 40 (29%) 18 (13%)
(2 hours)
Table 6 shows that DMAP can be used in the nuclear transfer procedure
along with other activation stimuli such as room temperature activation
(RTC) to[H increase developmental rates to the blastocyst stage in aged
oocytes (42 hour oocytes). RTC has been shown to be similar to an
electrical pulse or ionomycin activation stimulus (Stice and Keefer,
1992). EXPERIMENT 7
Use of Parthenogenically Activated Young Oocytes in a Nuclear Transfer
Procedure
This experiment was conducted under similar conditions as Experiment 6
with the exception that the oocytes were either placed directly in DMAP
(young oocytes) or given an ionomycin (5 mu M) treatment for 4 minutes
plus DMAP treatment immediately after enucleation, i.e., at 20 hours
maturation. At the end of the DMAP treatment, approximately 24 hours,
embryonic cells were transferred into the oocytes and electrofusion was
induced. The concentration ofactivating chemicals used were the same as
those described in Experiment 6. The embryos were placed in maturation
medium CR1aa for 6 to 7 days to determine developmental rates to
blastocyst stage. The results of this experiment are found in the
following Table 7:
TABLE 7
Development of 20-hours oocytes derived nuclear[H
transfers (fused after activation)
* * Development
Number Cleavage Rate to %
Treatment Oocytes Rate Blastocyst Usable
4 Hours 60 4% 0% 0%
DMAP
(Serum)
Ionomycin & 273 65% 24% 18%
4 hours
DMAP (BSA &
Serum)<1>
n<1> Activation Rate For Control Oocytes (BSA & Serum) 77/177 (44%) -
EXPERIMENT 8
Use of 24-hour Parthenogenically Activated Oocytes for Nuclear Transfer
Cumulus cells were removed from bovine oocytes at 21-22 hours maturation
by vortexing the oocytes in 200 mu l TL-HEPES for approximately 3 minutes.
The cells were then enucleated according to the procedures described in
Prather et al. Enucleation was verified by staining with 10 mg/ml Hoechst
33342, a DNA stain, for approximately 20 minutes, removing excess dye and
visualization with ultra-violet (UV) light (excitation emission). Oocytes
with no evidence of metaphase plate (enucleated oocyte) were selected.
The enucleated oocytes were exposed to 5 mu M ionomycin in TL-HEPES for
approximately 4 minutes. The enucleated oocytes were then diluted in
TL-HEPES (30 mg/ml bovine serum albumin [BSA]), and then diluted in
TL-HEPES (1 mg/ml BSA) .
The oocytes were then placed into maturation medium CR1 containing 1.9
mM DMAP for approximately 3.5-5 hours. Following the DMAP step, the
enucleated oocytes were diluted in TL-HEPES (1 mg/ml BSA) and a
blastomere, e.g., a donatednucleus, was inserted next to the oocyte.
The blastomere was electrofused to the enucleated oocyte according to
procedures described in Prather et al. at 100o V for about 30 mu secs. in
a 1 mmchamber. The electrofused nuclear transfer was incubated in
maturation medium CR1aa (1 mg/ml BSA) for 6 to 7 days. On day 4, 5%
heat-treated fetal calf[H erum was added.
The results are shown on the following Table 8:
TABLE 8
Use of 24-hour oocytes for nuclear transfer
using activation by Ionomycin (5 mu M) + 6-DMAP (1.9 mM)
Ionomycin + DMAP Control<1>
R<2> n<3> % +/- SE R n % +/- SE
Enucleation 11 68 91.2 +/- 3.0 * *
Fusion 11 443 91.8 +/- 1.5 8 141 90.3 +/- 2.2
>/= 2 Cell(d 2) 12 428 86.0 +/- 2.0 8 127 46.1 +/- 6.9
BL (d 6-7) 11 384 31.5 +/- 3.7 7 109 5.7 +/- 1.6
n<1> Nuclear transfer units not exposed to ionomycin or DMAP but fused at
similar oocyte age as the treated oocytes. -
[H
formed -
n<3> N = Number of units evaluated -
Following incubation in DMAP, the oocytes are diluted in TL-HEPES (1 mg/ml
BSA) and incubated in CR1aa (1 mg/ml BSA).
It is understood that the invention is not confined to the particular
construction and arrangement herein described, but embraces such modified
forms thereof as come within the scope of the following claims.
REFERENCES
Collas, P. and J. M. Robl. 1990. "Factors affecting the efficiency of
nucleartransplantation in the rabbit embryo." Biol. Reprod. 43:877-884.
Cuthbertson, K. S. R. 1983. "Parthenogenic activation of mouse oocytes in
vitro with ethanol and benzyl alcohol." J. Exp. Zool. 226:311-314.
First, N. L., Leibfried-Rutledge, M. L., Northey, D. L. and Nuttleman,
P. R. 1992. "Use of in vitro matured oocytes 24 h of age in bovine nuclear
transfer." Theriogenology 37:211.[H
First,N. L. and R. Prather. 1991. "Genomic Potential in Mammals,"
Differentiation, 48:1-8.
Fissore, R. A. and J. M. Robl. 1992. "Intracellular Ca<2 + > response of
rabbit oocytes to electrical stimulation." Mol. Reprod. Dev. 32:9-16.
Graham, C. F. 1969. "The fusion of cells with one and two cell mouse
embryos," Wistar Inot. Symp. Monogr., 9:19.
Graham, C. F. 1970. "Parthenogic mouse blastocysts." Nature 242:475-476.
Graham, C. F. 1974. "The production of parthenogenic mammalian embryos and
their use in biological research." Biol. Rev. 49:399-422.
Gray, K. R., K. R. Bondioli and C. L. Betts. 1991. "The commercial
application of embryo splitting in beef cattle." Theriogenology 35:37-45.
Kaufman, M. H. 1981. "Parthenogenesis: a system facilitating
understanding offactors that influence early mammalian development. Prog.
in Anat., Vol. 1, 1-34.
[H nd the role of calcium in exocytosis and cell
cycle activation in the mouse egg." Dev. Biol.149:80-89.
Masui, Y. and Markert, C. L. 1971. "Cytoplasmic control of nuclear behavior
during meiotic maturation of frog oocytes." J. Exp. Zool. 177, 129-146.
Nagai, T. 1987. "Parthenogenic activation of cattle follicular oocytes in
vitro with ethanol." Gamete Res. 16:243-249.
Nurse, P. 1990. "Universal control mechanism regulating onset of M-phase."
Nature 344:503-508.
Onodera, M. and Y. Tsunoda. 1989. "Parthenogenetic activation of mouse and
rabbit eggs by electric stimulation in vitro." Gamete Research 22:277-283.
Ozil, J. P. 1990. "The parthenogenetic development of rabbit oocytes after
repetitive pulsatile electrical stimulation." Development 109:117-127.
Prather et al., L. 1992. "Electrofusion-induced intracellular
Ca< + 2 > flux and its effect on murine oocyte activation." Mol. Reprod. Dev.
152-159.
Rosenkrans, U.S. Pat. No. 5,096,822.
Siracusa, G., Whittingham, D. G., Molinaro, M. and Vivarelli, E. 1978.
"Parthenogenic activation of mouse oocytes induced by inhibitors of protein
synthesis." J. Embryol. exp. Morph. 43:157-166.
Sirard, M. A., J. J. Parrish, C. B. Ware, M. L. Leibried-Rutledge and
N. L. First. 1988. "The culture of bovine oocytes to obtain
developmentally competent embryos." Biol. Reprod. 39:546-552.
Steinhardt, R. A., Epel, D. and Yanagimachi, R. 1974. "Is calcium
ionophore auniversal activator for unfertilized eggs?" Nature 252:41-43.
Stice, [0m S. L. and [7m Robl, [0m J. M. 1990. "Activation of
mammalian oocytes by a factor obtained from rabbit sperm." Mol. Reprod.
Dev. 25:272-280.
Stice, S. L. and Keefer, C. 1992. "Improved developmental rates for
bovine nucleus transfer embryos using cold shock activated oocytes." Biol.
Reprod. 42[H
(Suppl 1):166.
Surani, M. A. H. and Kaufmann, N. H. 1977. "Influence of extracellular
Ca<2 +> and Mg<2 + > ions on the second meiotic division of mouse oocytes:
Relevance to obtaining haploid and diploid parthenogenetic embryos." Dev.
Biol. 59:86-90.
Susko-Parrish, J. L., Nuttleman, P. R. and Leibfried-Rutledge, M. L.
1991. "Effect of bovine oocyte aging in vitro on development." Biol.
Reprod. 44(Suppl 1):17:156 (abstract).
Swann, K. 1990. "A cytosolic sperm factor stimulates repetitive calcium
increases and mimics fertilization in hamster eggs." Development 110:1295-1302.
Tarkowski, A. K., Witkowska, A. and Nowicka, J. 1970. "Experimental
parthenogenesis in the mouse." Nature 226:162-165.
Tarkowski, A. K. 1975. "Recent studies on parthenogenesis in the
mouse." In: The Developmental Biology of Reproduction (C. L. Markert, E.
J. Papaconstantinon, eds.), pp. 1071 varies as 129, New York: Academic
Press.
Tombes, R. M., Simerly, C., Borisy, G. G. and Schatten, G. 1992.
"Meiosis, egg activation and nuclear envelope breakdown are differentially
reliant on Ca< + 2> , whereas germinal vesicle breakdown is Ca< + 2 >
independent in the mouse oocyte." J. Cell Biol. 117:799-811.
Ware, C. B., Barnes, F. L., Maiki-Laurila, M. and First, N. L. 1989. "Age
dependence of bovine oocyte activation." Gamete Res. 22:265-275.
Watanabe, N., Hunt, T., Ikawa, Y. and Sagata, N. 1989. "Specific
proteolysis of the c-mos proto-oncogene product by calpain on
fertilization of Xenopus eggs." Nature 342:505-510.
Watanabe, N., Hunt, T., Ikawa, Y. and Sagata, N. 1991. "Independent
inactivation of MPF and cytostatic factor (mos) upon fertilization of Xenopus
eggs." Nature 352:247-248.
Whitaker, M. and Patel, R. 1990. "Calcium and cell cycle control."
Development 108:525-542.
Whitaker, M. and Irvine, F. R. 1984. "Inositol 1,4,5-trisphophate
microinjection activates sea urchin eggs." Nature (London) 312:636-639.
Yang, X., Jiang, S. and Shi, Z. 1992. "Improved activation by combined
cycloheximide and electric pulse treatment of bovine follicular oocytes[H
ured in vitro for 23-24 hours." Biol. Reprod. 42(Suppl 1):117.
CLAIMS: We claim:
[*1] 1. A process for the in vitro parthenogenic activation of a bovine
oocyte comprising the following steps in sequence:
a. increasing intracellular levels of divalent cations in the oocyte; and
b. reducing phosphorylation of cellular proteins in the oocyte.
[*2] 2. The process of claim 1 wherein the intracellular levels of
divalentcation are increased by introducing a divalent cation into the
oocyte cytoplasm.
[*3] 3. The process of claim 2 wherein the divalent cation is selected
fromthe group consisting of magnesium, strontium, barium and calcium.
[*4] 4. The process of claim 2 wherein the divalent cation is a free
calcium ion.
[*5] 5. The process of claim 2 wherein the divalent cation is
introduced byan ionophore.[H
[*6] 6. The process of claim 5 wherein the ionophore
is selected from the group consisting of ionomycin and A23187.
[*7] 7. The process of claim 1 wherein the intracellular levels of
divalentcation are increased by electric shock.
[*8] 8. The process of claim 1 wherein the intracellular levels of
divalentcation are increased by treatment with ethanol.
[*9] 9. The process of claim 1 wherein the intracellular levels of
divalentcation are increased by treatment with caged chelators.
[*10] 10. The process of claim 1 wherein phosphorylation of cellular
proteins is reduced by inhibiting phosphorylation.
[*11] 11. The process of claim 10 wherein the process of inhibiting
phosphorylation comprises adding an effective phosphorylation inhibiting
amount of a serine-threonine kinase inhibitor to the oocyte.
[*12] 12. The process of claim 11 wherein the serine-threonine kinase
inhibitor is selected from the group consisting of 6-dimethylaminopurine,
staurosporine, 2-aminopurine, and sphingosine.[H
f claim 11 wherein the serine-threonine kinase
inhibitor is 6-dimethylaminopurine.
[*14] 14. The process of claim 1 wherein phosphorylation of cellular
proteins is reduced by inducing dephosphorylation in the oocyte.
[*15] 15. The process of claim 1 wherein the oocyte is a 10-52 hour
oocyte.
[*16] 16. The process of claim 1 wherein the oocyte is a 16-30 hour
oocyte.
[*17] 17. The process of claim 1 wherein the oocyte is is approximately
24 hours old.
[*18] 18. A process for the in vitro parthenogenic activation of a 10-52
hour bovine oocyte comprising the following steps in sequence:
a. increasing intracellular levels of divalent cation in the oocyte by
introducing a divalent cation into the oocyte cytoplasm; and
b. reducing phosphorylation of cellular proteins in the oocyte wherein
phosphorylation of cellular proteins is reduced by adding a serine-threonine
kinase inhibitor to the oocyte in an amount effective to inhibit[H
ation.
[*19] 19. The process of claim 18 wherein the divalent cation is selected
from the group consisting of magnesium, strontium, barium and calcium.
[*20] 20. The process of claim 18 wherein the divalent cation is a free
calcium ion.
[*21] 21. The process of claim 18 wherein the serine-threonine kinase
inhibitor is 6-dimethylaminopurine.
[*22] 22. The process of claim 18 wherein the oocyte is a 16-30 hour
oocyte.
[*23] 23. The process of claim 18, wherein the oocyte is is approximately
24 hours old.
[*24] 24. A process for the in vitro parthenogenic activation of a bovine
oocyte comprising the following steps in sequence:
a. increasing intracellular levels of divalent cation in the oocyte;
and
b. reducing phosphorylation of cellular proteins in the oocyte by
introducinga dephosphorylizing amount of a phosphatase to the oocyte.
[*25] 25. The process of claim 24 wherein the phosphatase is selected
from the group consisting of Phosphatase 2A and Phosphatase 2B.
[H
5,453,366
Sep. 26, 1995
Method of cloning bovine embryos
INVENTOR: Sims, Michele M., 10955 FM 3356, Anna, Texas 75409
Rosenkrans, Jr., Charles F., 3169 Misa, Fayetteville, Arkansas 72703
ASSIGNEE-AFTER-ISSUE: Date Transaction Recorded: Feb. 03, 1997
ASSIGNMENT OF ASSIGNOR'S INTEREST (SEE DOCUMENT FOR DETAILS).
ABS GLOBAL, INC. 6908 RIVER ROAD DEFOREST, WISCONSIN 53532
Reel & Frame Number: 8328/0318
APPL-N0: 31,815
FILED: Mar. 15, 1993
REL-US-DATA:
Continuation-in-part of Ser. No. 606,418, Oct. 31, 1990 now abandoned Which
is a continuation-in-part of Ser. No. 558,969, Jul. 26, 1990 now patented
5,096,822[H
US-CL: 435#172.3; 800#DIG4; 800#DIG6; 800#2;
CL: 435;800;
SEARCH-FLD: 800#2; 435#172.3
REF-CITED:
U.S. PATENT DOCUMENTS
[7m <=1> [0m 4,994,384 2/1991 * Prather et al.
[7m <=2> [0m 5,057,420 10/1991 * Massey
[7m <=3> [0m 5,096,822 3/1992 * Rosenkrans
OTHER PUBLICATIONS
Bavister et al., 1983, "Development of Preimplantation Embryos of the Golden
Hamster in a Defined Culture Medium, " Biol. Reprod., 28:235.
Bondioli, K. R. et a.. 1990, "Production of Identical Bovine Offspring by
Nuclear Transfer," Theriogenology, vol. 13(1):165-174.[H
2, "Transplantation of Living Nuclei from Blastula Cells
into Enucleated Frogs' Eggs, " Zoology 38:455-463.
Briggs, R., et al., 1964, "Transplantation of Nuclei of Various Cell Types from Neurulae of the Mexican Axolotl (Ambystoma mexicanum)," Develop. Biol. 10:233.
Comandon and de Fonbrune, 1939, "Greffe Nucleaire Totale, Simple ou Multiple,
Chez une Amibe," Soc. Biol. 130:744.
Critzer, E. S. and N. L. First, 1986, "Use of Flourescent Stain for
Visualization of Nuclear Material in Living Oocytes and Early Embryos," Stain
Technology, 61(1) :1-5.
Eyestone, et al., 1987, "Culture of One-and Two-Cell Bovine Embryos to the
Blastocyst Stage in the Ovine Oviduct", Theriogenology, 28:1-7.
Elsdale et al., 1960, "A Description of the Technique for Nuclear
Transplantation in Xenopus laevis," J. Embryol. Exp. Morph., 8(4):437-444.
First and Prather, 1991, "Genomic Potential in Mammals," Diffeerentiation,
48:1-8.
Graham, C. F., 1969, "The Fusion of Cells with One and Two Cell Mouse Embryos," Wistar Inot. Symp. Monogr., 9:19.
Illmensee, K. and P. C. Hoppe, 1981, "Nuclear Transplantation in Mus musculus:
Development Potential of Nuclei from Preimplantation Embryos," Cell 23:9.
Kono, T., et al., 1991, "Development of Enucleated Mouse Oocytes Reconstituted
with Embryonic Nuclei," J. Reprod. Fert., 93:165-172.
McGrath, J. and D. Solter, 1983, "Nuclear Transplantation in the Mouse Embryo[H
by Microsurgery and Cell Fusion," Science, 220:1300.
McGrath, J. and D. Solter, 1984, "Inability of Mouse Blastomere Nuclei
Transferred to Enucleated Zygotes to Support Development In Vitro," Science,
226:1317-1319, 1984.
Navara, C. S. et al., 1992, "Timing of Polarization in Bovine Embryos and
Developmental Potential of Polarized Blastomeres," (Abstract) Proc. Soc. Study
Reprod., North Carolina State University (Jul. 12-15, 1992), p. 71.
Parrish, J. J. et al., 1988, "Capacitation of Bovine Sperm by Heparin," Biol.
Reprod. 38, 1171-1180.
Prather, R. S. et al., 1987, Biol. Reprod., 37:859-866.
Prather, R. S. et al., 1989, Biol. Reprod., 41:414-418.
Robl, J. M. et al., 1986, "Nuclear Transplantation in Mouse Embryos: Assessment of Recipient Cell Stage," Biol. Reprod., 34:733-739.
Sirard et al., 1988, Biol. Reprod., 39:546-552.
Smith, L. C. and I. Wilmut, 1989, "Influence of Nuclear and Cytoplasmic Activityon the Development in vivo of Sheep Embryos after Nuclear Transplantation,"
Biol. Reprod., 40:1027-1035.
[7m Stice, [0m S. L., and J. M. [7m Robl, [0m 1988, Biol. Reprod., 39:657-664.
Surani, M.A.H. et al., 1986, "Nuclear Transplantation in the Mouse: Heritable
Differences Between Paternal Genomes after Activation ofthe Embryonic Genome."
Cell, 45:127-136.
Tsunoda, Y. et al., 1987, J. Exp. Zool., (242:147-151).[H
986, Nature, 320:63-65.
PRIM-EXMR: Stone, Jacqueline
ASST-EXMR: Crouch, Deborah
LEGAL-REP: Dewitt Ross & Stevens
ABST:
An improved method of cloning mammalian embryos by transferring a nucleus
from a donor mammalian embryo to an enucleated recipient oocyte and culturing
the nuclear transferred bovine embryo in vitro is described. The improvement
includes positioning the donor embryo nucleus adjacent to the enucleated
recipient oocyte, and maintaining the nucleus and oocyte in a maintenance mediumfor a time sufficient to mature the cytoplasm of the enucleated recipient oocyteprior to fusing the donor nucleus to the recipient oocyte.
NO-OF-CLAIMS: 3
EXMPL-CLAIM: [7m <=4> [0m 1
[H
NO-DRWNG-PP: 0
PARCASE:
REFERENCE TO RELATED APPLICATION
This is a continuation-in-part of U.S. Ser. No 07/606,418, filed on Oct. 31, 1990, entitled "METHOD OF CLONING MAMMALIAN EMBRYOS," abandoned which is a
continuation-in-part of Ser. No. 558,969 filed Jul. 26, 1990 now U.S. Pat. No.
[7m <=5> [0m 5,096,822, which application and patent are incorporated herein by
reference.
SUM:
FIELD OF THE INVENTION
The present invention is generally directed to an improved method for cloningor multiplying mammalian embryos and to an improved method for transferring the nuclei of donor embryos into enucleated recipient oocytes, that is, oocytes fromwhich the nucle
i have been removed. The present invention is specifically
directed to enhancing mammalian embryo development after nuclear transfer by[H
including a maintenance period prior to fusion which promoted increased inner
cell mass surface area contact.
DESCRIPTION OF THE PRIOR ART
Advanced genetic improvement and selection techniques continue to be sought
in the field of animal husbandry. With specific reference to dairy cattle, for
example, significant increases in milk production have been made with the wide
scale use of genetically superior sires and artificial insemination. Dairy cows today produce nearly twice as much milk as they did 30 years ago. Further
genetic improvement can be accomplished by the multiplication of superior or
genetically manipulated embryos by cloning.
It has now become an accepted practice to transplant embryos in cattle to aidin the production of genetically superior stock. The cloning of embryos togetherwith the ability to transplant the cloned embryos makes it possible to produce
multiple genetically identical animals. Embryo cloning is the process of
transferring the nucleus of an embryonic donor cell to an enucleated recipient
ovum or oocyte. The clone then develops into a genetically identical offspring
to the donor embryo.
Nuclear transfer was first accomplished in Amoeba sphaeronucleus in 1939 by
Comandon and de Fonbrune. This was followed in 1952 by successful nuclear[H
ransfer in Rana pipiens by Briggs and King. The procedure for successful
nuclear transfers, according to Briggs and King (1952), included the following:
1) activation of a recipient oocyte:
2) enucleation, i.e., the process of removing or inactivating the chromosomesfrom the recipient oocyte; and
3) transfer of a whole blastomere (a cell resulting from embryo cleavage
prior to gastrulation) with a nucleus, from a blastula or early gastrula stage
embryo back to the enucleated oocyte.
Embryo cloning has now been successfully performed in cattle, sheep, rabbits,pigs and mice (Prather, et al., 1987; Willadsen, 1986; [7m Stice and Robl, [0m 1989;
Prather, et al., 1989; Tsunoda, et al., 1987).
Elsdale, et al. (1960) utilized ultraviolet irradiation to, in one step,
inactivate the egg pronucleus and activate the unfertilized oocyte. In the
axolotl, activation was reported by electrical shock with chromosomes of the eggnucleus being eliminated by ultraviolet irradiation, (Briggs, et al., 1964).
Transfer of a whole blastomere containing a nucleus into the enucleated oocyte
via a small bore micropipette was the common method of nuclear transfer for[H
all these techniques.
Two techniques have been used for nuclear transfer in the mouse. Illmensee
and Hoppe (1981) used a totally surgical method in which a micropipette was
inserted through the plasma membrane and into the cytoplasm of a pronuclear
stage embryo for pronuclear removal and subsequent insertion of donor nuclei.
McGrath and Solter (1983) reported a nondisruptive method of transplanting
nuclei. Nuclei were removed as membrane bounded pronuclear karyoplasts without
penetrating the plasma membrane of the embryo. The nucleus was inserted into a
recipient cell by cell fusion, using Sendai virus as the fusigenic agent. A
small volume of Sendai virus suspension was aspirated after removal of the donornucleus and the virus suspension and the pronuclear karyoplasts were injected
sequentially into the perivitelline space of the recipient embryo. At best, the microsurgical method of Illmensee and Hoppe (1981) was about 30-40% efficient,
whereas the nondisruptive method of McGrath and Solter (1983) was greater than
90% efficient. These techniques have been successful in producing blastocyst
stage embryos which do not continue development to term. Reports that Illmensee and Hoppe produced three live mice have been questioned.
It was later reported that blastocyst stage embryos and mice were produced bytransferring nuclei into enucleated pronuclear zygotes only when the donor cell stage was also pronuclear or at a very early two-cell stage (McGrath and[H
Pat. No. 5453366, * Solter, 1984; Surani, et al., 1986; and Robl, et al., 1986).
While cloning procedures have been successful for a variety of species, the
embryo development after nuclear transfer is lower than non-manipulated
contemporaries (Bondioli, et al. 1990).
Embryo development after nuclear transfer has been improved by various
procedures involving arresting the embryo cytoskeleton with a cytochalasin B
culture during cell fusion. Cytochalasin causes the embryo cytoskeleton to
become more elastic, making the embryo more conducive to cell fusion (McGrath
and Solter, 1983). It is believed that periods of cytoskeletal repair before
cell fusion may increase subsequent embryo development.
SUMMARY OF THE INVENTION
The present invention is directed to a technique which improves known methodsof producing cloned mammalian embryos by transferring a nucleus from a donor
embryo to a recipient oocyte. The improved cloning method includes isolating a
donor membrane-bounded nucleus from a cell of a donor embryo, removing the
nuclear chromosomal material from an oocyte to create an enucleated recipient
oocyte, maintaining the donor membrane-bounded nucleus and the enucleated
recipient oocyte for a period of time sufficient to increase inner cell mass
surface area contact between the donor membrane-bounded nucleus and the[H
cleated recipient oocyte, and fusing the membranes of the donor
membrane-bounded nucleus and the enucleated recipient oocyte together to form anembryonic single cell with a nucleus from the donor embryo, referred to herein
as a "nuclear transfer embryo" or an "NT". The improvement in this invention
lies in the maintenance period prior to fusion.
The present invention is more specifically directed to a method of
transferring a donor membrane-bounded nucleus isolated from a donor bovine
embryo, the embryo being at a stage of development where significant cell
differentiation has not occurred, to a recipient metaphase II bovine oocyte and culturing the nuclear transferred embryo in vitro. The method comprises removingthe nuclear chromosomal material from an oocyte to form an enucleated recipient oocyte; maintai
ning the donor membrane-bounded nucleus and the enucleated
recipient oocyte in a maintenance medium for a period of time sufficient to
mature the cytoplasm, such that the matured cytoplasm enables the oocyte to
advance the nucleus directly into cleavage without decondensing or recondensing the chromatin matter in the cytoplasm; and fusing the membranes of the donor
membrane-bounded nucleus and the enucleated recipient oocyte together to form anembryonic single cell with a nucleus from the donor embryo.
The present invention is also directed to a method for producing cloned
mammalian embryos. The method comprises removing the nuclear chromosomal[H
terial from a metaphase II bovine oocyte to form an enucleated recipient
oocyte; maintaining the enucleated recipient oocyte in a maintenance medium for a period of time sufficient to mature the cytoplasm, such that the matured
cytoplasm enables the oocyte to advance the nucleus directly into cleavage
without decondensing or recondensing the chromatin matter in the cytoplasm; and inducing cell fusion between the membranes of the donor membrane-bounded nucleusand the enucleated recipient oocyte to form an embryonic single cell with a
nucleus from the donor.
The present invention is further directed to an improved method for producingcloned mammalian embryos by transferring a donor membrane-bounded nucleus
isolated from a donor bovine embryo, the embryo being at a stage of development where significant cell differentiation has not occurred, to an enucleated
recipient metaphase II bovine oocyte and culturing the nuclear transfer embryo
in vitro. The improvement comprises maintaining the enucleated recipient oocyte in a maintenance medium for a period of time sufficient to activate the
cytoplasm, such that the activated cytoplasm enables the oocyte to advance the
nucleus directly into cleavage without decondensing or recondensing the
chromatin matter in the cytoplasm.
Further still, the present invention is directed to a method of transferring a donor membrane-bounded nucleus isolated from a donor bovine embryo, the[H
embryo being at a stage of development where significant cell differentiation
has not occurred, to a recipient metaphase II bovine oocyte and culturing the
nuclear transferred embryo in vitro. The method comprises removing the nuclear
chromosomal material from an oocyte to form an enucleated recipient oocyte;
maintaining the enucleated recipient oocyte in a maintenance medium for
approximately 6-28 hours prior to fusing the cell membranes; and fusing the
membranes of the donor membrane-bounded nucleus and the enucleated recipient
oocyte together to form an embryonic single cell with a nucleus from the donor
embryo.
By maintaining the nucleus of the membrane-bounded donor embryo and the
enucleated recipient oocyte in a maintenance medium for an elongated period of
time, typically 24-52 hours after oocyte aspiration from ovarian follicles, the nucleus and oocyte undergo a physiological change which enhances the ability of each of the membranes to fuse. This results in increased fusion and
developmental rates.
Further objects, features and advantages of the present invention will be
apparent from the following detailed description and appended claims.
REFERENCE TO CITATIONS
[H
disclosure is found in the
Bibliography preceding the claims.
DETDESC:
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to an improvement in the series of steps
which collectively result in the cloning of mammalian embryos by nuclear
transplantation. Although it is contemplated that the embryo cloning procedure
of the present invention may be utilized on a variety of mammals, the procedure will be described with reference to the bovine species. However, the present
invention does not restrict the cloning procedure to bovine embryos.
The cloning procedure includes a non-disruptive method of removing the
nucleus from a mature recipient oocyte and isolating a nucleus from a donor
embryo, bounded by a membrane, either by removal of the nucleus from the donor
embryo or by isolating a blastomere itself. The nucleus is then positioned so itis adjacent to the recipient oocyte and the nucleus is fused with the recipient cell to form an embryonic single cell.
[H
No. 5453366, * The standard embryo cloning process follows a basic six step procedure:
1) selecting a proper recipient oocyte and a multicellular embryo donor for
nuclear transfer;
2) isolating a blastomere from the donor;
3) enucleating, i.e., removing the nuclear material from the recipient
oocyte;
4) introducing the membrane-bounded nucleus of the blastomere adjacent to theenucleated recipient oocyte;
5) orienting the nucleus and the recipient oocyte for cell fusion; and
6) fusing the membrane surrounding the nucleus to the membrane of the
recipient oocyte and activating the recipient oocyte by dielectrophoresis,
The overall procedure disclosed herein may be described as cloning or as
multiplication of embryos by nuclear transfer followed by a prolonged
maintenance period to increase fusion and developmental rates of multiple
genetically identical embryos, and ultimately, animals.[H
os
The donor embryos may be obtained by flushing from surgically recovered
oviducts or may be nonsurgically flushed from the uterus in manners known to theart.
Preferred donor embryos generally have a development of from 2 to 64 cells.
In domestic animals such as cattle, an embryo at the 32-cell stage is called a
morula. At this stage, the cells become more compacted together. After the
morula stage, an internal inner cell mass space enlarges to create a central
fluid-filled cavity known as the blastocoel. At this stage, the embryo is known as a blastocyst. The cells of the blastocyst form a spherical shell having an
outer cell layer known as the trophectoderm. One pole of the blastocyst is
distinguished by a thicker accumulation of cells, known as the inner cell mass
(ICM), inside the trophectoderm. The embryo is derived from the ICM, while the
cells of the trophectoderm are the precursor of extraembryonic tissues and the
placenta. Reference is made to Navara et al. (1992) for a more detailed
description of this development.
Donor embryos at the blastocyst stage of development can be used for cloning as long as significant cell differentiation has not occurred. In the ICM of a
later stage embryo, for example, the cells may remain totipotent; that is,[H
they may not have undergone significant cellular differentiation, whereas the
outer cells have differentiated to form the trophectoderm and are not suitable
for use in embryonic cloning processes (Navara et al., 1992).
For the morula stage, i.e., 32-60-cell stage, Navara et al. (1992) showed
totipotency in nonpolarized cells, but not in polarized cells. Polarization is
the first evidence of commitment to cellular differentiation for the first
differentiation event. The polarized cells are identified by membrane staining
of the microvilli surrounding the cells. ICM cells do not always have microvilliaround them, but the cleaving stages do. At the cleaving stages, if the
microvilli are stained entirely around the cell, then the cells are not
polarized. If the microvilli have patches of stain missing, they are polarized.
The cells of the ICM are known as embryonic stem (ES) cells because they havethe ability to proliferate in an undifferentiated state, as well as give rise todifferentiated products. The isolation and multiplication in culture of
totipotent embryonic stem cells have value in providing a large population of
identical cells for use by nuclear transfer to produce clonal offspring (First
and Prather, 1991). Nuclear transfer of nuclei from murine ICM into enucleated
oocytes was performed by Illmensee and Hoppe (1981) and Kono et al. (1991) with limited success. Smith and Wilmut (1989) disclosed the production of sheep
embryo by electrofusion-mediated nuclear transplantation in which uncultured[H
single cells derived from the ICM of early blastocyst stage embryos were fused
to unfertilized enucleated secondary oocytes.
In bovine embryo nuclear transfer processes, donor cells from the 2-cell
stage through the inner cell mass stage of the blastocyst have been shown to be totipotent. This includes cultured cells of the inner cell mass.
Methods of in vitro maturation, fertilization and development also can be
used to generate donor embryos. Donor embryos at the 16-64 cell stage are
sometimes referred to as morula rather than blastula. Nevertheless, for
convenience the term blastula will be used herein to refer to the embryo,
regardless of age, and the term blastomere will be used to refer to a single
cell from any such embryo.
The nucleus of the donor embryo should be membrane-bounded to be used
optimally in the procedure. Such a membrane-bounded nucleus may either consist
of an entire blastomere or may consist of a karyoplast, which is an aspirated
cellular subset including a nucleus and a small amount of cytoplasm bounded by aplasma membrane.
Micromanipulation of the embryos is performed in a manner similar to the
methods of McGrath and Solter (1983), which is incorporated herein for details[H
of the micromanipulation technique. Micromanipulation is performed using a cell holding pipette, having an outer diameter of about 120 to 180 micrometers and aninner diameter of approximately 25 to 35 micrometers, and a beveled, sharpened
enucleation and transfer pipette having an outer diameter of approximately 10 to45 micrometers, depending upon the size of the blastomere.
The donor embryos may optionally be treated with cytochalasin B. The nuclei
of the donor embryos are prepared for transfer either by aspirating a part of
the blastomere which contains the nucleus, thus creating a karyoplast, or by
aspirating the entire blastomere. Aspirating the entire blastomere is preferred.
Culture and Maintenance Media
There are a variety of embryo culture and maintenance media routinely used
for the collection of embryos, and specifically bovine embryos. Examples of
known media, which may be used for bovine embryo culture and maintenance,
include Ham's F-10 + 10% fetal calf serum, Tissue Culture Medium-199 (TCM 199) +10% fetal calf serum, Tyrodes's-Albumin-Lactate-Pyruvate, Dulbecco's Phosphate
Buffered Saline, Eagle's and Whitten's media. One of the most common media for
the collection and freezing of embryos is Dulbecco's Phosphate Buffered Saline
(PBS) incorporating 1 to 20% fetal calf serum, new born serum or steer serum. Ifthe embryos are to be kept in an embryo culture medium for a substantial[H
period of time, the PBS is normally supplemented with 10 to 20% serum.
Eyestone, et al. (1987) reported that ligated ovine oviducts would support
development of bovine embryos from the 1-cell to blastocyst stage. Pregnancies
and live calves were produced after transfer of cultured embryos to recipient
heifers. Cultures of 1- and 2-cell embryos in the oviducts of intact cycling,
ovariectomized or anestrous ewes produced morphologically normal morulae and
blastocysts followed by pregnancies in recipient heifers, suggesting that
ovarian activity was not required for normal embryo development in the oviduct.
Another embryo culture and maintenance medium is described in parent U.S.
Pat. No. [7m <=6> [0m 5,096,822 to Rosenkrans, Jr. et al., entitled "Bovine Embryo
Medium," which is incorporated herein by reference. This embryo medium, named
CR1, comprises a culture solution containing the nutritional substances
necessary to support an embryo and is the preferred maintenance medium for use
with this invention. The medium contains hemicalcium L-lactate in amounts
ranging from 1.0 mM to 10 mM, preferably 1.0 mM to 5.0 mM. Hemicalcium L-lactateis L-lactate with a hemicalcium salt incorporated thereon. Hemicalcium L-lactateis significant in that a single component satisfies two major requirements in
the culture medium: 1) the calcium requirement necessary for compaction and
cytoskeleton arrangement; and 2) the lactate requirement necessary for
metabolism and electron transport. Hemicalcium L-lactate also serves as[H
uable mineral and energy source for the medium necessary for viability of theembryos.
Advantageously, CR1 medium does not contain serum, such as fetal calf serum, and does not require the use of a co-culture of animal cells or other biologicalmedia, i.e, media comprising animal cells, e.g., oviductal cells. Biological
media can sometimes be disadvantageous in that they may contain trace factors
which may be harmful to the embryos and which are difficult to detect,
characterize and eliminate.
Examples of the main components in CR1 medium include hemicalcium L-lactate, sodium chloride, potassium chloride, sodium bicarbonate and a minor amount of
fatty-acid free bovine serum albumin. Additionally, a defined quantity of
essential and non-essential amino acids may be added to the medium.
Salts are added to the medium to maintain a proper osmotic pressure or
osmolarity of the medium. The preferred osmotic pressure is 265 milli-osmoles
(mOSM). The concentration of salt generally ranges from 0 Mm to 155 Mm,
preferably 110 mM to 115 mM. Examples of salts include sodium chloride and
potassium chloride, preferably sodium chloride.
[H
is added as a surfactant, i.e., to prevent the
embryos from adhering to each other. Unlike culture media known to the art, the CR1 medium requires only a minimal amount of the fatty-acid free BSA. The
fatty-acid free BSA is added in amounts ranging from 1 mg/ml to 6 mg/ml,
preferably 1 mg/ml to 3 mg/ml, and most preferably 3.0 mg/ml. Antibiotically
effective amounts of an agent, such as gentamicin sulfate, penicillin,
streptomycin, fungizone or other antibiotics, known to the art, can also be
added. Additionally, a cryopreservation agent, known to the art, may be added toprotect the cellular integrity of the bovine embryo during freezing operations. Further, chemical buffers, such as HEPES, may by added to maintain the proper pHin the absence
of CO2.
CR1 medium preferably contains the following components in the following
quantities:
sodium chloride 114.7 mM
potassium chloride 3.1 mM
sodium bicarbonate 26.2 mM
hemicalcium L-lactate 5 mm
fatty-acid free BSA 3 mg/ml
[H
The "oocyte," as used here for the recipient oocyte, means an oocyte which
develops from an oogonium and, following meiosis, becomes a mature ovum. It has been found that not all oocytes are equally optimal cells for efficient nuclear transplantation in mammals. For purposes of the present invention, metaphase II stage oocytes,
matured either in vivo or in vitro, have been found to be
optimal. Mature metaphase II oocytes may be collected surgically from either
nonsuperovulated or superovulated cows or heifers 35 to 48 hours past the onset of estrus or past an injection of human Chorionic Gonadotrophin (hCG) or similarhormone. Alternatively, immature oocytes may be recovered by aspiration from
ovarian follicles obtained from slaughtered cows or heifers and then may be
matured in vitro in a maturation medium by appropriate hormonal treatment and
culturing. As stated above, the oocyte is allowed to mature in a known
maturation medium until the oocyte enters the metaphase II stage, generally
18-24 hours post aspiration. For purposes of the present invention, this period of time is known as the "maturation period." As used herein for calculation of
time periods, "aspiration" refers to aspiration of the immature oocytes from
ovarian follicles.
Mature oocytes can be first treated with cytochalasin B at about 7.5
micrograms per milliliter, or an effectively similar microtubal inhibitor at a[H
concentration sufficient to allow the enucleation and transfer pipette to be
inserted through the zona pellucida to allow for removal of a portion of the
cytoplasm without, at any point, actually rupturing the plasma membrane. The
mature oocyte is first held in place by mild suction by the cell holding
pipette. The enucleation and transfer pipette is then inserted through the zona pellucida of the oocyte at the point of either the metaphase II bulge or
adjacent to the first polar body, i.e., in a location intended to be adjacent tothe metaphase chromosomes. The pipette does not penetrate the plasma membrane.
Aspiration applied through the pipette draws a portion of the cytoplasm into thepipette which includes, in the case of the metaphase II bulge, the entire bulge surrounding cytoplasm, or, in the case of the first polar body, the cytoplasm
adjacent to the polar body. This process is intended to draw all the metaphase
chromosomes into the pipette. As the pipette is withdrawn, with suction
maintained, the plasma membrane is stretched and then seals itself leaving a
competent plasma membrane on the enucleated oocyte.
Maintenance Period
Prior to fusing the membranes of the donor membrane-bounded nucleus and the
enucleated recipient oocyte together, it has been found that maintaining the
nucleus and the oocyte together in the embryo maintenance medium significantly
improves the development of the embryo post fusion. The enucleated oocyte is[H
allowed to remain in the maintenance medium for a period of time sufficient to
activate the cytoplasm, such that the activated cytoplasm enables the oocyte to advance the nucleus directly into cleavage without decondensing or recondensing the chromatin matter in the cytoplasm. The period is generally about 6-28 hours,preferably abou
t 10-18 hours, after the oocyte maturation period to align the
oocyte adjacent the membrane of the nucleus. Calculated from oocyte aspiration, the maintenance period will be about 24-52 hours, preferably 28-42 hours, post
aspiration.
It was originally believed that the maintenance period increased the inner
cell mass surface area contact area between the nucleus and the oocyte
It is now known that the enucleated recipient oocyte should be maintained in the maintenance medium for a period of time sufficient to mature the cytoplasm
in the oocyte, such that the matured state of the cytoplasm enables the oocyte
to advance the nucleus of the donor embryo directly into cleavage without
decondensing or recondensing the chromatin matte in the cytoplasm, after the
nucleus has fused to the recipient oocyte. Processing the donor nucleus is
affected by both age and enucleation status of the recipient oocyte.
The nucleus does not need to be in the maintenance medium during this time.
The recipient oocyte should, however, be allowed to remain the maintenance[H
medium for a period of time sufficient to mature the cytoplasm. Maturation of
the cytoplasm reduces the activity of the cytoplasm. Thus, the presence of the
oocyte in the medium will not prematurely decondense and recondense chromatin
material in the nucleus and the fused cell will progress to nuclear division.
The transfer pipette, carrying the aspirated membrane-bounded nucleus, is
inserted through the zona pellucida of the recipient enucleated oocyte; and the membrane-bounded nucleus is deposited under the zona pellucida with its membraneabutting the plasma membrane of the recipient oocyte.
Fusion of the Nucleus and the Oocyte
At approximately 24-52 hours, preferably 28-42 hours post aspiration or,
stated differently, 6-28 hours, preferably 10-18 hours, after the nucleus is
positioned such that its membrane is adjacent the enucleated oocyte, the cell
surface contact appears to be at its peak. It is on this basis that membrane
fusion may be enhanced with the use of the maintenance period of this invention.
Cell Fusion
A variety of fusion techniques may be employed for this invention. For
example, the onset of the electricity by electrofusion can induce the fusion[H
process. Electrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane. This
breakdown of the plasma membrane is very short and the membrane reforms very
rapidly. If two adjacent membranes are induced to breakdown and upon reformationthe lipid bilayers intermingle, small channels will open between the two cells. Due to the thermodynamic instability of such a small opening, it enlarges until the two cells b
ecome one. Reference is made to U.S. Pat. No. 4,994,384 to
Prather et al., which is incorporated herein by reference, for a further
discussion of this process. A variety of electrofusion media can be used
including sucrose, mannitol, sorbitol and phosphate buffer solution.
Fusion can also be accomplished using Sendai virus as a fusigenic agent
(Graham, 1969).
Polyethylene glycol (PEG) may also be used as a fusigenic agent. Under
prescribed conditions, PEG provides excellent fusion results. In one protocol,
the cells are fused in PEG (molecular weight 1,300-1,600 Sigma), which is mixed in a solution containing TL Hepes (approximately 1:0.25 mu g/ml) and polyvinyl
alcohol (PVA) (approximately 1 mu g/ml), CA<2 + > and MG<2 + > -free. The media containing the cells is then passed through one or more dilutions (approximately1:1) of the above-described PEG media. The cell media is then allowed to rest ina culture media
, such as TL Hepes containing fetal calf serum until the cell[H
urn to a normal appearance. To activate the embryos, the embryos
are washed in CA<2 + > and MG<2 + > -free TL Hepes and exposed to an ionophore, for example, Ionomycin (Calbiochem). This is followed by another rest in TL
Hepes containing fetal calf serum, after which the embryos are returned to a
maturation medium for further maturation. Experimental conditions will vary
depending upon the products used.
The techniques incorporated herein proved to be successful in producing
blastocysts that, when transferred to cattle, were capable of establishing
pregnancies.
In the preferred embodiment, fusion of the membrane-bounded nucleus with the enucleated recipient oocyte and simultaneous activation of the recipient oocyte are carried out by a single dielectrophoresis step using commercially available electrofusion e
quipment which is described below. Prior to electrofusing the
donor embryo nucleus and enucleated recipient oocyte together, it is necessary
to orient the cell membranes in the electric field. The term "orientation" as
used herein is defined as the placement of the two cells such that the plane of contact of the two membranes, i.e., the plasma membrane of the body carrying thedonor nucleus and the plasma membrane of the recipient oocyte, which will becomefused together,
is perpendicular to the electrical field. It has been found thatrandom orientation results in a marked reduction in the successful fusion[H
rate. If cells are oriented such that the fusion membranes are parallel, or at
approximately a 45o angle, to the electrical field, the rate of successful
fusion will decrease. The alignment may be done electrically or mechanically. Ifthe size of the two cells is not greatly disproportionate, a small alignment
alternating-current voltage ( approximately equal to 5 volts per millimeter at
1000 KHz) for a short time (10 seconds) will cause the cells to reorient with
their membranes apposed. Repeated pulses may be needed. If the cells vary
greatly in size, mechanical manipulation may be required to properly orient the membranes.
The actual incorporation of a donor nucleus into an enucleated oocyte is
conducted by a dielectrophoretic method of cell fusion, using a DC current and
using a non-conductive, i.e., non-ionic, media such as a mannitol solution,
sorbitol or sucrose (Zimmerman) based cell fusion media. The fusion phenomenon
is the result of cell membrane breakdown and pore formation between properly
oriented opposing cells. The pores, or small channels, created between the two
cells are thermodynamically unstable because of the high surface curvature of
the channels and the associated high tension in the membrane. This instability
causes the channels to merge and enlarge until the membranes form a single cell which contains the nucleus from the donor embryo cell.
[H
Fusion can be detected by staining techniques known to the art, for example
the Hoechst staining procedure (Critzer (1986), or by visual inspection.
Post-Fusion Culture
The embryonic single-cell clones produced as described herein preferably are cultured, either in vitro or in vivo, to the morula or blastula stage. For
example, the clones may be cultured in the oviduct of sheep, or other suitable
animal, or in a suitable culture medium. The embryos then may be transplanted
into the uteri of cattle at a suitable stage of the estrous cycle. The
procedures for transplantation are commonly known and practiced in the embryo
transfer field. A percentage of these transplants will initiate pregnancies in
the maternal surrogates. Animals born of these pregnancies will be genetically
identical where the donor cells were from a single embryo or a clone thereof.
The following example is offered by way of illustration and not by way of
limitation.
EXPERIMENTAL PROCEDURES
Source of Recipient Oocytes and Donor Embryos. The oocyte-cumulus complexes
(OCC) were aspirated from bovine ovaries recovered from a slaughterhouse in
Milwaukee, Wis. The ovaries were transported from the slaughterhouse to the[H
laboratory in insulated containers with saline (0.9%; 30o +/- 2o C.). After
arrival, the ovaries were washed with 30o C. water and placed into a flask
containing fresh saline (0.9%) in a water bath maintained at 30o C. Follicular
contents were harvested by aspiration using an 18-gauge needle and accumulated
in a 50 ml sterile conical tube (Falcon #2070). Following aspiration, OCC were
located using 100 mm plates in a warm room (30o +/- 2o C.) using a
stereomicroscope. The OCC were washed 4 times through low bicarbonate-TALP
(Parrish, et al. (1988), incorporated herein by reference, and placed into
maturation plates (10 OCC per 50 ul drop). Maturation plates consist of ten 50
ul drops of maturation medium with estradiol-17B (1 ug/ml), FSH (0.5 ug/ml) and LH (0.5 ug/ml) (Sirard et al., 1988) in a 60 mm petri dish with 10 ml of
paraffin oil.
Those oocytes not used as recipient oocytes were fertilized to provide donor embryos in subsequent experiments. Fertilization of oocytes was performed
according to the procedure described in Sirard et al. (1988). Briefly, OCC were in vitro matured 22 +/- 2 hours, then washed 2 times through Sp-TALP (Parrish,
et al., 1988) and moved into fertilization plates. Fertilization was performed
in 60 mm plates with 10 drops (40 mu l each) of fertilization medium (5 ml TL
Stock-no glucose, 30 mg fatty-free BSA-6 mg/ml final, 50 mu l pyruvate stock-0.2mM final, 2.5 mu l gentamicin-25 mu g/ml final-optional) covered with oil and 10OCC for each drop. The OCC were added, followed by 5 x 10<4 > motile sperm,[H
hypotaurine, 1 mu m epinephrine, 21 mu m sodium metabisulfate, and 118 mu m
DL-lactate syrup in 0.9 % sodium chloride according to Leibfried and Bavister,
1982).
Motile sperm cells were prepared by the Percoll separation of frozen sperm
procedure. A 45% percoll solution (3.090 ml/100 ml KCl, 2.920 ml/100 ml NaH2PO4,4.675 g/100 ml NaCl, 2.380 g/100 ml HEPES, adjust the pH to 7.3) was prepared bya 1:1 dilution with 90% percoll using Sperm TL Stock Solution (SPTL) (2.10 mM
CaCl2<2> H2O, 3.1 mM KCl, 0.4 mM MgCl26H2O, 100 mM NaCl, 0.29 mM NaH2PO4H2O,
21.6 mM Lactic Acid, 10 mM Hepes, 25 mM NaHCO3, adjust pH to 7.4 before bringingto final volume; check osmolarity: 290-300 mOSM) according to Parrish, et al.,
1988. The SPTL did not contain BSA. Two-three ml of 90% percoll was placed on
the bottom of a conical tube (Falcon 2095). Two ml of 45% percoll was layered
carefully on the top of the 90% percoll. The procedure was performed at room
temperature. One unit of semen was thawed (35o C., 1 min.) and layered on top ofthe percoll gradients. The tube was centrifuged at 700 x g for 30 min. The top
layers were removed, and the concentrated motile sperm was available for use.
Following 44 +/- 2 hours after insemination, the OCC were stripped of cumuluscells, and the eggs were categorized as cleaved ( >/= 2 cells) or uncleaved. Thecleaved eggs were cultured in CR1-L-Glutamine medium for 3 to 5 days until[H
Pat. No. 5453366, * they reach the morula or blastocyst stage.
Embryo Handling and Micromanipulation.
Recipient oocytes were enucleated by aspirating approximately one-eighth to
one-fourth the cytoplasm juxtaposed to the polar body or the metaphase bulge
using a 10-45 micron transfer pipette, leaving an enucleated membrane-bounded
oocyte. Embryos were manipulated in calcium and magnesium-free TL Hepes bufferedmodified Tyrodes medium prepared according to Bavister et al., 1983. Nuclei fromlater stage donor embryos were removed by aspirating the nucleus and some
surrounding membrane-bounded cytoplasm from a blastomere or by aspirating an
entire blastomere. Micromanipulation was conducted using a holding pipette
having an outer diameter of approximately 120-180 microns and an inner diameter of approximately 30 microns and a beveled, sharpened enucleation and transfer
pipette having an outer diameter of approximately 10 to 45 microns. Whole
blastomeres, containing nuclei, were removed from donor embryos and positioned
in the perivitelline space of the recipient oocytes by the method of McGrath andSolter (1983).
Maintenance Procedures
Embryos were maintained in vitro in CR1-L-Glutamine medium for 6-18 hours
prior to fusion. Zimmerman Cell Fusion Medium (GCA Corporation, Chicago,[H
l.), was used for fusing donor and recipient cells. Cells from donor embryos
were washed in the medium then placed in the fusion chamber with the Zimmerman
medium. Following the fusion treatment, oocytes were placed in CR1-L-Glutamine
medium, in 50 microliter drops, under paraffin oil in a humidified 5% CO2 in airincubator and monitored for fusion and allowed to develop for five days.
Activation and fusion of the intact, membrane-bounded nuclei to the
enucleated oocytes were carried out in Zimmerman Cell Fusion Medium by
dielectrophoresis using a Zimmerman Electrofusion Instrument, GCA Corporation,
Chicago, Ill. The fusion chamber consisted of two parallel electrodes 1 mm aparton a glass slide. The instrument was adjusted in the following manner:
Fusion voltage: 80-120 volts (DC)
Electrode distance: 1 mm
Alignment voltage: 1-5 volts (AC)
Alignment frequency: 1000 KHz
Pulse duration: 10-40 microseconds[H
ds
Number of Pulses: 1-6
Experiment
The experiment was designed to determine whether the development of nuclear
transfer embryos (NTs) could be improved by maintaining the donor embryo cells
and recipients in maintenance medium for an extended period of time. NTs were
produced according to Prather et al. (1987) which is incorporated herein by
reference. Specifically, in vitro matured oocytes were used as recipients, in
vitro developed embryos were used as donors, and the subsequently formed NTs
were developed in vitro in CR1 with 1 mM L-glutamine medium (CR1-L-GLN).
The treatment structure was a blocked one-way design with 3 treatments. Each donor embryo served as the block, i.e., every treatment was imposed on donor
embryo cells from each donor embryo. Enucleation and transfer of donor
blastomeres was performed at 24 hours post-oocyte aspiration. The three
treatments were as follows: 1) fusion at 30 hours; 2) those that did not fuse at30 hours and were then refused at 42 hours; and 3) fusion at 42 hours. During
the manipulation process, the donor and recipient embryo cells were handled in
calcium and magnesium free TALP-hepes. The cells were maintained in CR1-L-GLN
during the waiting period before fusion as well as is the post-fusion[H
opment period. Embryos were developed in 50 microliter drops of this
maintenance medium under paraffin oil in a humidified incubator with an
atmosphere of 5% CO2 in air. This experiment was replicated on three different
days using three morula stage donor embryos (30-40 cells each) on each day of
nuclear transfer. The results are illustrated in the following table:
Nuclear Transfer - Fusion at 30 Hours
Versus 42 hours
* * DEVELOPMENT
* * MORULA +
FUSION ACTIVATION CLEAVAGE BLASTOCYST
30 hr * *
N 59/104 68/97 19/59 2/59
% 56.9 71 32.0 4.7 +/- 3.7
30/42 hr
N 31/42 * 19/31 7/31
% 80.1 * 63.9 22.2 +/- 6.7
42 hr
N 87/108 80/99 62/87 28/87
% 80.4 81 71.0 32.6 +/- 5.3[H
> : cultured 5 days in CR1 + L-Glutamine (1 Mm) -
n : uncommon superscripts differ (P < 0.05) -
The results indicate that when the fusion regime is changed to a later time
period, i.e., following the maintenance period of this invention, development ofNTs is similar to that of normal in vitro matured, fertilized and developed
oocytes. Thus, the nuclear transfer embryo appears to have increased fusion and developmental rates when fusion is delayed by approximately 6-18 hours after thetime fusion would normally take place, i.e., 34-48 hours after aspiration of therecipient oocyt
es.
It is understood that the invention is not confined to the particular
construction and arrangement herein described, but embraces such modified forms thereof as come within the scope of the following claims.
BIBLIOGRAPHY
U.S. Pat. No. 4,994,384 to Prather et al.
U.S. Pat. No. 5,096,822 to Rosenkrans, Jr. et al.
[H
983, "Development of Preimplantation Embryos of the Golden Hamster in a Defined Culture Medium," Biol. Reprod., 28: 235.
Bondioli, K.R. et al. 1990, "Production of Identical Bovine Offspring by
Nuclear Transfer," Theriogenology, Vol. 13 (1): 165-174.
Briggs and King, 1952, "Transplantation of Living Nuclei from Blastula Cells into Enucleated Frogs' Eggs," Zoology 38:455-463.
Briggs, R. , et al. , 1964, "Transplantation of Nuclei of Various Cell Types from Neurulae of the Mexican Axolotl (Ambystoma mexicanum)," Develop. Biol 10:
233.
Comandon and de Fonbrune, 1939, "Greffe Nucleaire Totale, Simple ou Multiple,Chez une Amibe," Soc. Biol. 130:744.
Critzer, E. S. and N. L. First, 1986, "Use of Fluorescent Stain for
Visualization of Nuclear Material in Living Oocytes and Early Embryos," Stain
Technology, 61(1):1-5.
Eyestone, et al., 1987, "Culture of One- and Two-Cell Bovine Embryos to the
Blastocyst Stage in the Ovine Oviduct" Theriogenology, 28:1-7.[H
t al., 1960, "A Description of the Technique for Nuclear
Transplantation in Xenopus laevis," J. Embryol. Exp. Morph., 8(4):437-444.
First and Prather, 1991, "Genomic Potential in Mammals, " Differentiation,
48:1-8.
Graham, C. F., 1969, "The Fusion of Cells with One and Two Cell Mouse
Embryos," Wistar Inot. Symp. Monogr., 9:19.
Illmensee, K. and P. C. Hoppe, 1981, "Nuclear Transplantation in Mus
musculus: Development Potential of Nuclei from Preimplantation Embryos, " Cell
23:9.
Kono, T., et al., 1991, "Development of Enucleated Mouse Oocytes
Reconstituted with Embryonic Nuclei, " J. Reprod. Fert., 93:165-172.
McGrath, J. and D. Solter, 1983, "Nuclear Transplantation in the Mouse Embryoby Microsurgery and Cell Fusion," Science, 220:1300.
McGrath, J. and D. Solter, 1984, "Inability of Mouse Blastomere Nuclei
Transferred to Enucleated Zygotes to Support Development In Vitro," Science,
226:1317-1319, 1984.[H
in Bovine Embryos and
Developmental Potential of Polarized Blastomeres, " (Abstract) Proc Soc. Study
Reprod., North Carolina State University (Jul. 12-15, 1992), p. 71.
Parrish, J.J. et al., 1988, "Capacitation of Bovine Sperm by Heparin," Biol. Reprod. 38, 1171-1180.
Prather, R.S. et al., 1987, Biol. Reprod., 37:859-866.
Prather, R.S. et al., 1989, Biol. Reprod., 41:414-418.
Robl, J.M. et al., 1986, "Nuclear Transplantation in Mouse Embryos:
Assessment of Recipient Cell Stage," Biol. Reprod., 34:733-739.
Sirard et al., 1988, Biol. Reprod., 39:546-552.
Smith, L.C. and I. Wilmut, 1989, "Influence of Nuclear and Cytoplasmic
Activity on the Development in vivo of Sheep Embryos after Nuclear
Transplantation," Biol. Reprod., 40:1027-1035.
[7m Stice, [0m S.L., and J.M. [7m Robl, [0m 1989, Biol. Reprod., 39:657-664.[H
Surani, M.A.H. et al., 1986, "Nuclear Transplantation in the Mouse: HeritableDifferences Between Paternal Genomes after Activation of the Embryonic Genome." Cell, 45:127-136.
Tsunoda, Y.. et al., 1987, J. Exp. Zool., 242:147-151).
Willadsen, S.M., 1986, Nature, 320:63-65.
CLAIMS: What is claimed is:
[*1] 1. A method for producing cloned bovine embryos comprising the
following steps:
a. removing the chromosomal material from a metaphase II stage bovine oocyte to form an enucleated recipent oocyte;
b. maintaining the enucleated recipient oocyte in CR-1 + 1 mM L-glutamine
maintenance medium wherein the maintenance period is approximately 6 to 18
hours;
c. placing a membrane bound nucleus from a donor 2-64 cell bovine embryo
adjacent to the plasma membrane of the enucleated recipient oocyte and;[H
d. inducing cell fusion between the membranes of the donor membrane-bound
nucleus and the enucleated recipient oocyte to form an embryonic single cell
with a nucleus from the donor, wherein the membranes are either electrically
fused or fused with polyethylene glycol.
[*2] 2. The method of claim 1 wherein the CR-1 + 1 mM L-glutamine
maintenance media further comprises 5 mM hemicalcium L-lactage and 0.4 mM
pyruvate.
[*3] 3. The method of claim 1 further comprising, before step. d.,
orienting the donor membrane-bound nucleus and the enucleated recipient oocyte
so that the plane of contact of their membranes is perpendicular to the
direction of electric current flow.
[H
5,057,420
Oct. 15, 1991
Bovine nuclear transplantation
INVENTOR: Massey, Joseph M., College Station, Texas
ASSIGNEE-AT-ISSUE: Granada Biosciences, Inc., Houston, Texas (02)
ASSIGNEE-AFTER-ISSUE: Date Transaction Recorded: Nov. 14, 1991
SECURITY INTEREST (SEE DOCUMENT FOR DETAILS).
COOPERATIEVE CENTRALE RAIFFEISEN-BOERENLEENBANK B .A., "RABOBANK NEDERLAND" A
BANKING ORGANIZATION OF THE NETHERLANDS 245 PARK AVE., NEW YORK, NEW YORK
Reel & Frame Number: 5916/0955
Date Transaction Recorded: Feb. 24, 1992
ASSIGNMENT OF ASSIGNORS INTEREST.
W. R. GRACE & CO. - CONN., A CORP. OF CT 1 TOWN CENTER ROAD BOCA RATON, FL
33486-1010
Reel & Frame Number: 6036/0538[H
RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS).
GRANADA BIOSCIENCES, INC.
Reel & Frame Number: 6036/0543
Date Transaction Recorded: Feb. 01, 1995
SECURITY INTEREST (SEE DOCUMENT FOR DETAILS).
JOHN HANCOCK MUTUAL LIFE INSURANCE COMPANY 200 CLARENDON STREET BOSTON, MA 02116JOHN HANCOCK LIFE INSURANCE COMPANY OF AMERICA 200 CLARENDON STREET BOSTON, MA
02116
MASSACHUSETTS MUTUAL LIFE INSURANCE COMPANY 1295 STATE STREET SPRINGFIELD, MA
01111
MASSMUTUAL CORPORATE INVESTORS 1295 STATE STREET SPRINGFIELD, MA 01111
MASSMUTUAL PARTICIPATION INVESTORS 1295 STATE STREET SPRINGFIELD, MA 01111
Reel & Frame Number: 7320/0109
Date Transaction Recorded: Feb. 01, 1995
SECURITY INTEREST (SEE DOCUMENT FOR DETAILS).
JOHN HANCOCK MUTUAL LIFE INSURANCE COMPANY 200 CLARENDON STREET BOSTON, MA 02116MASSACHUSETTS MUTUAL LIFE INSURANCE COMPANY 1295 STATE STREET SPRINGFIELD, MA
01111
MASSMUTUAL CORPORATE INVESTORS 1295 STATE STREET SPRINGFIELD, MA 01111[H
MUTUAL PARTICIPATION INVESTORS 1295 STATE STREET SPRINGFIELD, MA 01111
Reel & Frame Number: 7320/0166
Date Transaction Recorded: Mar. 15, 1995
ASSIGNMENT OF ASSIGNOR'S INTEREST (SEE DOCUMENT FOR DETAILS).
ABS GLOBAL, INC. 7908 RIVER RD. DEFOREST, WI 53552
Reel & Frame Number: 7365/0442
APPL-N0: 599,256
FILED: Oct. 16, 1990
REL-US-DATA:
Continuation of Ser. No. 58,904, Jun. 5, 1987 now abandoned
INT-CL: [5] C12N 15#00
US-CL: 435#172.2; 435#172.3; 800#2; 800#DIG6; 935#53; 935#111
CL: 435;800;935
[H
935#53, 111
REF-CITED:
U.S. PATENT DOCUMENTS
[7m <=1> [0m 4,664,097 5/1987 * McGrath et al. 128#1R
FOREIGN PATENT DOCUMENTS
2199845 7/1988 * United Kingdom
OTHER PUBLICATIONS
McGrath et al., Science 220:1300-1302 (1983).
Lohse et al., Theriogenology 23(1):205 (1985).
Loskutoff et al., Theriogenology 25(1):168 (1986).
"Nuclear Transplantation in the Bovine Embryo: Assessment of Donor Nuclei and
Recipient Oocyte", R. S. Prather et al, Biology of Reproduction, vol. 37,
859-866 (1987).
Transplantation of Living Nuclei from Blastula Cells into Enucleated Frogs[H
Eggs, R. Briggs and T. J. King, Proceedings of the National Academy of Sciences,vol. 38, pp. 455-463 (1952).
Adult Frogs Derived from the Nuclei of Single Somatic Cells, J. B. Gurdon,
Developmental Biology, vol. 4, pp. 256-273 (1962).
Development and Chromosomal Constitution of Nuclear-Transplants Derived from
Male Germ Cells, M. A. DiBerardino and N. J. Hoffner, Journal of Experimental
Zoology, vol. 176, pp. 61-72 (1971).
Activation of Dormant Genes in Specialized Cells, M. A. DiBerardino, N. J.
Hoffner and L. D. Etkin, Science, vol. 224, pp. 946-952 (Jun. 1984).
Methods and Success of Nuclear Transplantation in Mammals, A. McClaren, Nature, vol. 309, pp. 671-672, (Jun. 21, 1984).
Nuclear Transplantation in Mouse Embryos, J. McGrath and D. Solter, The Journal of Experimental Zoology, vol. 228, pp. 355-362 (1983).
Development of Reconstituted Mouse Eggs Suggests Imprinting of the Genome DuringGametogensis, M. A. H. Surani, S. C. Barton, M. L. Norris, Nature, vol. 308, pp.548-550 (Apr. 5, 1984).
Full-Term Development after Transplantation of Parthenogenetic Embryonic Nuclei into Fertilized Mouse Eggs, P. C. Hoppe, K. Illmensee, Proceedings of the
National Academy of Sciences, vol. 79, pp. 1912-1916 (Mar. 1982).
Nuclear Transplantation in Mus musculus: Developmental Potential of Nuclei from Preimplantation Embryos, K. Illmensee, P. C. Hoppe, Cell, vol. 23, pp. 9-18
(Jan. 1981).[H
. Vienken, Journalof Membrane Biology, vol. 67, pp. 165-182 (1982).
Multiplication of Bovine Embryos, R. S. Prather, F. L. Barnes, J. M. Robl and N.L. First, Society for the Study of Reproduction, Ithaca, New York (1986) Biologyof Reproduction, vol. 34, Supplement 1, p. 192, Jun. 4, 1986.
Nuclear Transplantation in Bovine Embryos, J. M. Robl, R. Prather, W. Eyestone, F. Barnes, D. Northey, B. Billigan and N. F. First, Theriogenology, vol. 25, No.1, p. 189 (Jan. 1986).
Nuclear Activation and Transplantation in the Rabbit Oocyte, S. L. [7m Stice, [0m H.
M. Bean, J. M. [7m Robl, [0m Biology of Reproduction, Supplement 1, p. 77 (1987);
presented Society for the Study of Reproduction; 20th Anniversary Meeting,
Urbana, Illinois (Jul. 1987).
Electrofusion for the Pronuclear Transplantation of Mouse Eggs, Y. Tsunoda, Y.
Kato and Y. Schioda, Gamete Research, vol. 27, No. 1, p. 209 (Jan. 1987).
Multiplication of Bovine Embryos, F. L. Barnes, R. S. Prather, J. M. Robl and N.L. First, Theriogenology, vol. 27, No. 1, p. 209, Jan. 1987.
Nuclear Transplantation in Bovine Embryos, J. M. Robl, R. Prather, E. Barnes, W.Eyestone, D. Northey, B. Gilligan and N. L. First, Journal of Animal Science,
vol. 64, pp. 642-647 (1987).
Nuclear Transplantation in Mouse Embryos: Assessment of Nuclear Function, F. L. Barnes, J. M. Robl and N. L. First, Biology of Reproduction, vol. 36, pp.
1267-1274 (1987).[H
, Nature, vol. 320, No.6057, pp. 63-65, Mar. 6, 1986.
PRIM-EXMR: Weimar, Elizabeth C.
ASST-EXMR: Chambers, Jasemine C.
LEGAL-REP: Boulware; Margaret A.
ABST:
A process to produce viable bovine embryos through nuclear transplantation.
The process utilizes nuclear material from 4 cell and later stage developed
embryos recovered from inseminated donor cows through nonsurgical or surgical
means. Recipient eggs or oocytes are recovered and a portion of the ooplasm
removed by micromanipulation. Individually separated cells from the nuclear
material donor embryo are inserted into the recipient oocyte. The oocyte
fragment and nuclear donor material are electrofused. The embryo is cultured in vitro, transferred for an interim time to a sheep oviduct and then transferred
to a cow for gestation.
NO-OF-CLAIMS: 20[H
NO-OF-FIGURES: 0
NO-DRWNG-PP: 0
PARCASE:
This application is a continuation of co-pending application Ser. No.
058,904, filed June 5, 1987, now abandoned.
SUM:
BACKGROUND OF THE INVENTION
Nuclear transfer involves the transplantation of living nuclei from typicallyembryonic cells to unfertilized eggs. The early research on vertebrates was
performed in amphibians. The embryonic frog blastomere cells were separated and the nuclear material was introduced into frogs' eggs which had been enucleated. See Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs
Eggs, R. Briggs and T. J. King, Proceedings of the National Academy of Sciences,Volume 38, pages 455-463, 1952. Further experimentation was performed on[H
amphibians and amphibian eggs to determine if nuclear material from adult frog
somatic or germinal cells could be transplanted to eggs and develop into a
normal larva. Development and Chromosomal Constitution of Nuclear-Transplants
Derived from Male Germ Cells, M. A. Berardino and N. Hoffner, Journal of
Experimental Zoology, Volume 176, pages 61-72, 1971. There is a great degree of uncertainty as to when the genetic material of a cell can no longer be
reprogrammed, which limits the stage of development of donor nuclear material.
Transplantation of nuclear material in mammals has proved very difficult to
achieve which is in part due to the microsurgical techniques on mammalian
embryos and eggs. The microsurgical techniques can be destructive to delicate
cell structure which damages the cell material used in the later stage of the
transplantation procedure. An alternative procedure is to deliver the nuclear
material to a recipient egg by fusion of an intact cell or a karyoplast
consisting of a nucleus surrounded by a piece of plasma membrane to the egg. Themanipulation to isolate a karyoplast is performed in the presence of
cytochalasin B. Methods and Success of Nuclear Transplantation in Mammals, A.
McLaren Nature, Volume 109, June 21, 1984. Also, once the nuclear material has
been transplanted to a recipient a mammalian egg there is fusion of the cellularmaterial to produce a new viable embryo. The fusion can be aided or induced withvirus or electro-field induced. However, the conditions for fusion are not
predictable. Electric Field-Induced Cell-to-Cell Fusion, U. Zimmermann and J.[H
Vienke, Journal of Membrane Biology, Volume 67, page 165-182 (1982).
Nuclear transplantation in higher mammals has been attempted. Successful
nuclear transplantation and cell fusion was achieved for sheep embryos when
individual blastomeres from 8 and 16 cell embryos were used as the nuclear
donors into enucleated or nucleated halves of unfertilized eggs. Nuclear
Transplantation in Sheep Embryos, S. M. Willadsen, Nature, Volume 320, pages
63-65, March 1986. Nuclear transplantation has been attempted in bovine embryos,however, the embryos developed only 43 days out of a nine month typical
gestation period. Nuclear Transplantation in Bovine, J. M. Robl, R. Prather, W. Eyestone, F. Barnes, D. Northey, B. Gilligan, and N. L. First, Theriogenology,
Volume 25, No. 1, January 1986. Successful nuclear transplantation and embryo
development in higher mammals has great implications in breeding.
SUMMARY OF THE INVENTION
Selective cattle breeding involves the selection of the desired bull as the
sperm donor and the desired female for the egg donor. Typically the female was
artificially inseminated with the bull semen in a selective breeding program.
The female may be superovulated to produce several eggs and therefor several
embryos. The embryos may then be transferred to recipient cows for gestation.
[H
This invention is a method for producing a relatively large number of
identical bovine embryos that can be transferred to surrogate female cows for
gestation. The process involves the separation of a donor embryo into single
cells which were transplanted into nucleated or enucleated oocytes for further
development. The donor embryo can be from a 4 to at least a compacted morula
stage embryo (approximately 64 cells). The earlier stage embryos must be removedsurgically. However, the 16 cell to compacted morula cell stage embryos can be
removed nonsurgically which is a much more economical method. For commercial
breeding application the embryos used as nuclear donors are selected from cows
that have been inseminated with the selected bull semen to produce the desired
cross or an embryo developed from a prior nuclear transfer. However, this
process may be used with any bovine multicellular embryo. Also, good results
have been obtained with previously deep frozen embryos.
The stage of the donor embryo at the 16 cell to compacted morula stage of
development makes the process attractive from an economical standpoint because
of the increased number of donor nuclear cells as well as the lack of surgical
technique necessary to remove the embryo initially. The process, whether or not a surgical procedure is used to remove embryos, enables one to prepare embryo
clones with a good success rate in viability. Although there were some losses inviability of the embryos during the early stage of development, this process
will produce multiple calves with identical genetic makeup.[H
s that were developed through this procedure have in turn been
used as nuclear donor cells for subsequent recipient eggs. This produces viable cattle embryos by transplantation of nuclear material from embryos which are
themselves the products of nuclear transplantation.
DETDESC:
DESCRIPTION OF THE PREFERRED METHOD
The process constitutes several steps which generally includes isolating the recipient oocytes from the donor cows, recovering embryos from donor cows,
transferring the nuclear material from the embryo to the recipient oocytes,
fusing the oocyte fragment and the donor cells to form an embryo, and culturing the embryo before transferring into a recipient cow for gestation. The recipientoocytes are collected from cows which have been induced to ovulate at a
predicted time with prostaglandin F2 alpha (pgF2 alpha ). Human chorionic
gonadotropin (hCG) can also be administered to further predict the ovulation
time. The oocytes were collected approximately 39 hours after hCG or 87 hours
after pgF2 alpha administration. Approximately 4,000 I.U. of hCG were
administered. If desired, the cow can be superovulated to produce multiple eggs by administering FSH hormone. The oocytes were collected surgically
approximately 39 hours after hCG administration. This is approximately 10 to[H
14 hours after ovulation.
The zona pellucida of the egg was cleaned of all cells and debris with a
cleaning pipette. The eggs were placed in a solution of Dulbecco's Phosphate
Buffed Saline (PBS). After cleaning the eggs were placed on a micromanipulation stage. Using a fine glass needle, a slit was made in the zona pellucida directlyover the polar body traversing the perivitelline space such that the zona
pellucida is opened slightly more than 180o along its equator with the polar
body near the middle of the slit. If the polar body was not located, a slit of
the same dimension was made in the part of the zona pellucida which spans the
widest portion of the perivitelline space.
After the recipient oocytes have been slit, they were placed in PBS
containing Cytochalasin B (5 micrograms per/ml; Sigma) for at least half an hourbefore further processing.
The nucleus donor embryo is typically collected from a cow which has been
artificially inseminated, however this procedure may be used with any cow embryofrom the 4 cell to compacted morula stage of development. For embryo donors fromthe 16 cell to compacted morula stage, nonsurgical procedures are used to flush the embryo from
the cow uterus. The donor embryos are collected after four and
half to six days after behavioral estrus is observed. The embryos are examined[H
after collection to determine the stage of development. In some cases the embryonuclear donor was from deep frozen embryos. Typically 16 to 32 cell embryos are collected on day five. Also late morulae and early blastocysts collected on day six have be
en used.
The zona pellucida of the nucleus donor embryo was slit with a fine glass
needle 3/4 or more around the equator. The embryo was coaxed out of the zona
pellucida with the tip of the needle and transferred to a petri dish with fresh PBS solution. The embryo was separated into blastomere cells by repeated suctionin a micropipette. An alternative procedure is to remove the individual cells
from the embryo by drawing the cells one at a time into a micropipette with an
inner diameter approximately the same size as the cells.
Returning to the oocytes which have been previously slit, if the polar body
was clearly visible, the polar body and an adjacent part of the ooplasm
corresponding to between 1/4 and 1/8 of the total volume of the cell was sucked into a micropipette. In oocytes with no visible polar body, about 1/2 of the
ooplasm was removed by suction with a micropipette. The ooplasm which was
removed can be inserted into an evacuated zona pellucida. The foreign zona
pellucida can be obtained from an egg that is not chosen as a recipient oocyte. Thus, one egg can yield two recipient oocytes if a foreign zona pellucida is
used.[H
tte to
pierce the zona pellucida. A micropipette was inserted in the egg and about halfof ooplasm was aspirated. The zona pellucida encloses the remaining ooplasm of
the donor egg and the ooplasm removed with the micropipette was placed in a
recipient zona pellucida.
The enucleated half of the egg, the half without the polar body, is presumed to fuse with the nuclear material that comes from the separated embryo cells.
However, sometimes it is not possible to identify the polar body so it was
difficult to determine where the nuclear material lies. It has been postulated
that an egg with nuclear material should not be viable with additional nuclear
material from the embryo cell. There is sometimes more than a 50% survival rate when one egg is divided and nuclear material transplanted into both halves. Thissupports the premise that possibly the nuclear material was broken up to a
degree that it is not viable. However, other theories could support the
simultaneous viable development of both egg halves.
The disassociated blastomeres of the donor embryo were transferred to the
halved recipient oocytes. A blastomere was transferred to the perivitelline
space and brought in close contact with the oocyte fragment. The
blastomere/oocyte fragments were then ready for fusion. The preferred method of fusion is electrofusion.[H
The embryos were placed in PBS at room temperature for one hour before
electrofusion. The zonae pellucidae containing blastomere and egg halves were
transferred to a petri dish containing fusion medium of 0.3M Mannitol, 0.1 mM
MgSO4, 0.05 mM CaCl2 in distilled water or Zimmerman cell fusion media for
approximately 25-30 minutes.
The blastomere/oocytes were transferred in the fusion medium to the chamber
of an electrofusion apparatus (Zimmermann; GCA, Chicago). With the Zimmerman
electrofusion apparatus the two electrical poles are wires which are parallel toeach other in the center of the plate. The oocyte with the blastomere are lined up at right angles to either wire such that the oocyte touches the wire and the blastomere port
ion is opposite to the wire. The blastomere/oocytes are exposed
to the following fusion conditions; cell alignment (600 kHz, 6 V for 10
seconds), followed by 3 fusion pulses of 15 V with a pulse duration of 50.0
microseconds at 0.1 second interval. After the fusion pulses, the alignment
voltage was reduced over one minute from 6 V to 0 V. All embryos were
transferred to PBS and incubated at 37o C. for one hour. The embryos are then
examined to determine in which fusion had occurred or is in progress.
A semi-solid medium such as agar can be used to embed the embryos prior to
transfer to sheep oviducts. Several embryos can be embedded in one section of
agar. The agar chip allows for normal development to occur in the ewe's[H
duct even though the zonae pellucidae are not intact.
An agar solution was prepared with 0.6 g agar dissolved in 60 ml of 0.9% NaClin distilled water by boiling. The agar solution was set aside to cool. The
embryos were transferred to a PBS solution with 20% sheep or calf serum. When
the agar solution has cooled to 30o to 35o C. the embryos are picked up in a
small amount of PBS medium with a micropipette. Five to 10 ml of agar solution
was poured into an empty petri dish and the embryos are transferred into the
agar using the pipette. The agar containing the embryo was then placed in a
petri dish containing PBS. The embryo in the agar is expelled from the pipette
shaped as a small cylinder which is cut short as possible.
A second embedding takes place using a 0.6 gm agar dissolved in 50 ml of 0.9%NaCl in distilled water by boiling. The agar solution was cooled to 30o to 35o
C. The first embedded embryos were completely covered. The second embedding withagar formed a chip not longer than 2.5 to 3 mm.
The agar embedded embryos were transferred into ligated oviducts of ewe for
culture. Four to six days later the oviduct ewe was flushed with PBS solution
which is inspected for the chip with the embedded embryo. The chip was cut open so that the cavity around the embryo is open and the embryo is dislodged with
glass needles. After release from the agar chip the embryos were placed in[H
fresh PBS at room temperature until they can be transferred to recipient cows ina conventional way.
The nucleus donor embryo used in this procedure can be a embryo which was
produced from the nuclear transplantation procedure of this invention. This
serial use of the nuclear material involves the use of nucleus donor embryo
prepared following the steps of the above described procedure. The embryo
recovered from the sheep is at a stage of development which can be used as a
nucleus donor. The embryo cells are separated and fused with recipient oocytes
as described above. The use of nuclear donor embryos prepared by nuclear
transplantation can be performed serially such that nuclear material can be fromseveral generations of embryonic material prepared by the procedures described
herein.
The embryos recovered from the sheep may be deep frozen. At a later time the embryos are thawed and used in the procedure as described herein.
The following Tables I and II give the results of the nuclear transplantationof bovine embryo cells to oocytes as described by this procedure. Table I is a
summary of first generation transfers in which the embryo used as the nucleus
donor was from an artificially inseminated cow. Table II is a summary of second generation transfer in which the embryo used as the nucleus donor was from a[H
first generation embryo developed by the nuclear transplantation process.
TABLE I
BOVINE NUCLEAR TRANSPLANTATION 1ST GENERATION
(GGI)
* No. No. No. * * No. * * No. No. Attempte Trans Tran
Embryo Donor d Fusion . No. Viable s. No. * Live Succes Recover Embryo Pregnan Aborti
Stage Embryos Fusion sful Sheep ed s Cow t ons Calves
16-20
cell 8 115 98 101 81 29 23 12 4 8 21-32
cell 14 326 261 290 256 71 63 14<1> 2 4 > 32
cell 6 145 121 125 107 26 25 4<2> 1 0 TOTAL 28 586 480 516 444 126 111 30 7 12[H
n<1> Fourteen transfers are pending pregnancy examination -
n<2> Four transfers are pending pregnancy examination -
TABLE II
BOVINE NUCLEAR TRANSPLANTATION SECOND GENERATION
* No. No. No. * * No. * * No. No. Attempte Trans Tran
Embryo Donor d Fusion . No. Viable s. No. * Live Succes Recover Embryo Pregnan Aborti
Stage Embryos Fusion sful Sheep ed s Cow t ons Calves
> 32
cell 4 83 58 55 51 31 22 3 2 1
As shown in Table I, 16 cell stage embryos and higher were used. The highest cell stage tested was compacted morula of about 64 cells. The lower cell stage
embryo nucleus donors were traditionally thought to be more suitable for
transfer of nuclear material. However, Table I shows a good fusion and[H
ility rate of the higher cell stage embryo compared to the lower cell stage.
The number of successful fusions are noted on both Table I and Table II. Thisfigure was calculated based on visual observation of the embryos under a
microscope after electrofusion. The number of successful fusions was rated by
visual observation. Some of the embryos which did not pass the visual test for
fusion were nevertheless transferred to the sheep for further culture.
Not all the embryos were carried through all the stages of the process as
shown in the Tables. Live calves were born from the process of this invention
demonstrating the success of the methods described.
Table II shows the use of second generation embryos, those developed by this invention, in subsequent nuclear transplantation. The embryos recovered from thesheep are used as nuclear material donors. Consequently, a descending number of generations can
be cloned using the same genetic material using the steps of
this transplantation process.
CLAIMS: What is claimed is:
[*1] 1. A process to produce viable bovine embryos through nuclear
transplantation comprising the following steps:[H
oocytes from donor cows;
(b) collecting embryos greater than 32 cells;
(c) removing a portion of the ooplasm from the recipient oocyte;
(d) separating the embryo cells into single donor cells for nuclear
transplantation;
(e) inserting said donor cell into the recipient oocyte;
(f) electrofusing the oocyte and donor cell to form an embryo; and
(g) culturing said embryo.
[*2] 2. A process to produce viable bovine embryos of claim 1 wherein the
embryos collected in step (b) are deep frozen and thawed for use later in the
process described in claim 1.
[*3] 3. A process to produce viable bovine embryos of claim 1 wherein in
step (c) the portion of the ooplasm removed is corresponding to at least 1/8 of the total volume of the cell and is adjacent to and includes the polar body.[H
[*4] 4. A process to produce viable bovine embryos of claim 1 wherein in
step (c) about half of the ooplasm is removed comprises the additional steps;
preparing a foreign zona pellucida to receive said ooplasm;
inserting said removed ooplasm into said foreign zone pellucida; and
following steps (d) through (e) of claim 1 utilizing the recipient oocyte andthe zona pellucida with transferred ooplasm as a recipient oocyte.
[*5] 5. A process to produce viable bovine embryos of claim 1 including thestep of transferring the cultured embryo to a recipient cow for gestation.
[*6] 6. A process to produce viable bovine embryos of claim 1 including thesteps of transferring the electrofused embryo to a surrogate animal for
culturing and subsequent transfer to a recipient cow for gestation.
[*7] 7. A process to produce viable bovine embryos of claim 1 including thestep wherein the electrofused embryo is embedded in a semi-solid medium; and
transferring the embedded embryo into a surrogate animal for culturing.[H
[*8] 8. A process to produce viable bovine embryos of claim 1 wherein said recipient oocytes are collected approximately 10 to 14 hours post ovulation fromthe donor cows.
[*9] 9. A process to produce viable bovine embryos of claim 1 wherein said nuclear donor embryos are at least 33 cells.
[*10] 10. A process to produce viable bovine embryos of claim 1 wherein
said recipient oocytes are collected approximately 39 hours after hCG
administration.
[*11] 11. A process to produce viable bovine embryos of claim 1 wherein
said recipient oocytes are collected approximately 87 hours after pgF2 alpha
administration.
[*12] 12. A process to produce viable bovine embryos through nuclear
transplantation comprising the following steps:
(a) collecting oocytes from donor cows;
(b) collecting embryos greater than 32 cells;[H
ellucida of at least one oocyte more than 180o;
(d) removing the polar body and at least 150 of the ooplasm of the oocyte;
(e) separating the embryo cells into disassociated blastomeres;
(f) inserting said blastomeres into the perivitelline space in close contact with the remaining ooplasm of the oocyte fragment;
(g) electro fusing the oocyte fragment and the blastomere to form an embryo; and
(h) culturing said embryo.
[*13] 13. A process to produce viable bovine embryos of claim 12 including the step of transferring the cultured embryo to a recipient cow for gestation.
[*14] 14. A process to produce viable bovine embryos of claim 12 including the steps of transferring the electrofused embryo to a surrogate animal for
culturing and subsequent transfer to a recipient cow for gestation.
[H
5] 15. A process to produce viable bovine embryos of claim 12 including the step wherein the electrofused embryo is embedded in a semi-solid medium; and
transferring the embedded embryo into a surrogate animal for culturing.
[*16] 16. A process to produce viable bovine embryos of claim 12 wherein
said recipient oocytes are collected approximately 10 to 14 hours post ovulationfrom the donor cows.
[*17] 17. A process to produce viable bovine embryos of claim 12 wherein
said nuclear donor embryos are at least 33 cells.
[*18] 18. A process to produce viable bovine embryos of claim 12 wherein
said embryos used for donor cells have been deep frozen and subsequently thawed prior to step (d).
[*19] 19. A process to produce viable bovine embryos of claim 12 wherein
said recipient oocytes are collected approximately 39 hours after hCG
administration.
[*20] 20. A process to produce viable bovine embryos of claim 12 wherein
said recipient oocytes are collected approximately 87 hours after pgF2 alpha[H
administration.
[H
You have pressed the ENTER
page of the last PATENT for this search level.
or FIRST PAGE key.[H