WO2001030970A2

WO2001030970A2 - Improved protocol for activation of oocytes

Info

Publication number: WO2001030970A2
Application number: PCT/US2000/029552
Authority: WO
Inventors: Jose Cibelli
Original assignee: Advanced Cell Technology, Inc.
Priority date: 1999-10-27
Filing date: 2000-10-27
Publication date: 2001-05-03
Also published as: AU1235401A; WO2001030970A3

Abstract

A novel protocol for activation of mammalian oocytes or nuclear transfer fusions that use a CDC2 or CDK2 specific inhibitor selected from roscovitine, iso-olomoucine, olomoucine, flavopiridol, and DMAP is provided. This protocol provides for more efficient formation of blastocysts which can be used to produce cloned offspring and embryonic stem cells.

Description

IMPROVED PROTOCOL FOR ACTIVATION OF OOCYTES

FIELD OF THE INVENTION

The present invention relates to an improved procedure for activation of oocytes.

The resultant activated oocytes may be used in nuclear transfer procedures for the

production of cloned embryos and offspring, and the production of embryonic stem cells.

BACKGROUND OF THE INVENTION

In the past several years significant attention in the press, both popular and

scientific, has been focused in the area of nuclear transfer and the production of cloned

embryos and offspring. Nuclear transfer recently gained significant worldwide attention

with the Roslin Institute's report of the production of a cloned sheep, Dolly, by

introduction of quiescent (non-dividing cells) into an enucleated ovine oocyte. (Wilmut

et al, Nature, 1385:810-813, "Viable Offspring Derived from Fetal and Adult Mammalian

Cells"). Shortly thereafter, a group of scientists at the University of Massachusetts and

Advanced Cell Technology, including the inventor of this application, reported the

production of three cloned, transgenic cows by nuclear transfer (Cibelli et al, Science,

280:1256 (1998)) using transgenic fetal fibroblast donor cells.

Moreover, recently a Japanese group reported the production of cloned cattle using

oviduct cells collected from an adult cow at slaughter. (New Scientist, July 11, 1998,

Hadlield et a, "Premature birth repeats the Dolly mixture'-;. Also, Jean-Paul Renard from INRA in France reported the production of a cloned calf using muscle cells obtained from

a fetus. (Mackenzie et al, New Scientist, March 11, 1998, "A French calf answers some

of the questions about cloning.") Further, David Wells from New Zealand recently

reported the production of a cloned calf using fibroblast cells obtained from an adult cow.

(Wells, D. N., "Cloning Symposium: Reprogramming Cell Fetal-Transgenesis and

Cloning", Monash Medical Center, Melbourne, Australia, April 15-16, 1998.)

Also, recently, a group at the University of Hawaii reported the production of

cloned mice and cloned progeny by nuclear transfer using cumulus cell donor nuclei

obtained from adult mice. (Wakagama et al, Nature, 394:369-379, 1998, "Full-term

development of mice from enucleated oocytes injected with cumulus cell nuclei".)

Thus, there have been reported many recent successes in the area of nuclear

transfer and cloning. With respect thereto, the efficacy of nuclear transfer and the

production of cloned embryos and offspring is reportedly affected by various factors

including the availability of suitable donor oocytes. In particular, such methods require

the availability of oocytes that are in a state of "activation."

Activation is a process that generally involves the elevation of intracellular

calcium in the egg. The sperm normally produces oscillations in calcium concentration

that last for several hours. Artificial activation protocols have been used on eggs for

many years. Early work indicated that ethanol, electrical shock, cooling, calcium-free

media, various anesthetics and a variety of other stimuli could cause activation. (Whittingham, D.G., 1980, ^Parthenogenesis in mammals", Oxford Rev. Reprod, Biol,

2:205-231) In more recent years, with the development of procedures for measuring

intracellular calcium and the various intracellular responses to calcium, more specific

approaches have been developed. For example, we now know that electrical pulses cause

transient increases in intracellular calcium by inducing pores in the membrane and

allowing calcium to flood into the cell from the extracellular media. (Fissore, R.A. and

Robl, J. M., 1992, "Intracellular calcium response of rabbit oocytes to electrical

stimulation", Mol. Reprod. Devel, 32:9-16; Collas, P., J.J. Balise, G.A. Hofrnan and

Robl, J.M., 1989, "Electrical activation of mouse oocytes", Theriogeneology, 32:835-844;

Robl, J. M., P. Collas, R. Fissore and JR. Dobrinsky, 1992, "Electrically induced fusion

and activation in nuclear transplant embryos", In: Guide to Electroporation and

Electrofusion; D. Chang, B.M. Chassy, J.A. Saunders and A.E. Sowers (ed.), Academic

Press, Inc., San Diego, CA; Collas, P., R. Fissore, J. M. Robl, E.J. Sullivan and F.L.

Barnes, 1993, "Electrically-induced calcium elevation, activation and parthenogenetic

development of bovine oocytes", Mol. Reprod. Devel, 34:212-223; Collas, P., R. Fissore

and J. M. Robl, 1993, "Preparation of nuclear transplant embryos by electroporation",

Anal. Bioc em., 208:1-9) Multiple pulses can be used to duplicate sperm-induced

calcium oscillations. Injection of such intracellular second messengers such as IP3, or

its long acting analogues, GTP, or its long acting analogues, or calcium itself can

duplicate sperm-induced calcium rises. Other compounds that cause calcium rises, although less physiological, are ethanol and calcium ionophores. (Fissore, R.A. and Robl,

J.M., 1993, "Sperm, inositol triphosphate and thimerosal induced intracellular Ca2+

elevations in rabbit eggs", Devel. Biol, 159:122-130; Fissore, R.A. and Robl, J.M., 1994,

"Mechanism of calcium oscillations in fertilized rabbits eggs", Devel. Biol, 166:634-642;

Fissore, R.A., Pinto-Correia, C. and J.M. Robl, 1995, "Inositol triphosphate-induced

calcium release in the generation of calcium oscillations in bovine eggs", Biol Reprod.,

53:166-11 A; Collas, P., Chang, T., Long, C. and J.M. Robl, 1995, "Inactivation of histone

HI kinase by Ca²⁺ in rabbit oocytes", Mol. Reprod. Devel, 40:253-258.) The second part

of the activation event is a decrease in a cell cycle regulatory kinase called MPF. This

results in a decrease in the phosphorylation of many different proteins in the cell and the

progression to interphase. This part of the process can be duplicated by various kinase

inhibitors. MPF can be inactivated directly by inhibiting protein synthesis and

compounds like puromycin and cycloheximide have been used successfully in oocyte

activation protocols. Currently, there are a number of different combinations of the above

that are being used successfully for oocyte activation in various laboratories around the

world.

A brief overview of known oocyte activation procedures are summarized in tabular

form below:

Barnes, F. L., Collas, P., Powell, R., King, W. A., Westhusin, M., and Shepherd, D. (1993). "Influence of Recipient Oocyte Cell Cycle Stage on DNA Synthesis, Nuclear Envelope Breakdown, Chromosome Constitution, and Development in Nuclear Transplant Bovine Embryos." Molecular Reproduction and Development(36), 33-41.

Campbell, K. H. S., Ritchie, W. A., and Wilmut, I. (1993). "Nuclear-Cytoplasmic Interactions during the First Cell Cycle of Nuclear Transfer Reconstructed Bovine Embryos: Implications for Deoxyribonucleic Acid Replication and Development." Biology of Reproduction^), 933-942.

Ectors, F.J., Delval, A., Smith, L. C, Touati, K., Remy, B., Beckers, J.-F., and Ectors, F. (1995). "Viability of Cloned Bovine Embryos After One or Two Cycles of Nuclear Transfer and In Vitro Culture." Theriogenology (44), 925-933.

Keefer, C L , Stice, S. L. and λ atthews, D. L. (1994). "Bovine Inner Cell Mass Cells as Donor Nuclei in the Production of Nuclear Transfer Embryos and Calves." Biology ofReproduction(50), 935-939. Lavoir, M.-C, Rumph, N., Moens, A., King, W. A., Plante, Y., Johnson, W. H., Ding, J., and Betteridge, K. J. (1997). "Development of Bovine Nuclear Transfer Embryos Made with Oogonia." Biology of Reproduction^ 6), 194-199.

Takano H., K. C, Kato Y, Tsunoda Y. (1996). "Cloning of bovine embryos by multiple nuclear transfer." Theriogenology, Al, 1365-1373.

Westhusin, M. E., Collas, P., Marek, D., Sullivan, E., Stepp, P., Pryor, J., and Barnes, F. (1996). "Reducing the Amount of Cytoplasm Available for Early Embryonic Development Decreases the Quality But Not Quantity of Embryos Produced by In Vitro Fertilization and Nuclear Transplantation." Theriogenology(A6), 243-252.

Zakhartchenko, V., Wolf, E., Palma, G. A., and Brem, G. (I 995). "Effect of Donor Embryo Cell Number and Cell Size on the Efficiency of Bovine Embryo Cloning." Molecular Reproduction and Developmental), 53-57.

Based on the foregoing, it is apparent that many different methods for producing

activated oocytes suitable for nuclear transfer are known. However, it would be

beneficial if more efficient methods for producing activated oocytes suitable for nuclear

transfer procedures could be developed. This would especially be useful in instances

wherein oocytes suitable for use in nuclear transfer are in short supply.

BRIEF DESCRIPTION AND OBJECTS OF THE INVENTION

Toward that end, it is an object of the invention to provide an improved method

for producing activated mammalian oocytes suitable for use in nuclear transfer

procedures.

More specifically, it is an object of the invention to provide an improved method

for producing activated ungulate oocytes, preferably activated bovine oocytes, for use in

nuclear transfer procedures. It is a further object of the invention to use the activated mammalian oocytes

produced by the subject method for the production of cloned embryos and offspring and,

more preferably, transgenic cloned embryos and offspring, and for the production of

embryonic stem cells.

Thus, the present invention relates to an improved method for effecting activation

of mammalian oocytes, preferably ungulate oocytes and, most preferably, bovine oocytes,

that results in efficient blastocyst development. This method is in particular an

improvement over the activation protocol reported by Susko-Parrish (Susko-Parrish J. L.,

Northey, David L, Leibfried-Rutledge, Mr. Lorraine; Stice, Steven L. (1996),

"Parthenogenic Oocyte Activation, U.S. Patent 5,496,370; Susko-Parrish, J. L., Leibfried-

Rutledge, M. L., Northey, D. L., D. L. Schutzkus, V., and First, N. L. (1994).)

DEFINITIONS AND DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Prior to describing the present invention in detail, the following terms are defined.

Unless otherwise defined, all terms in this application will have their ordinary accepted

scientific meanings.

Nuclear transfer: In the present invention, this refers to a procedure where a donor

cell or nucleus is inserted or injected into an enucleated oocyte (oocyte wherein the

nucleus has been removed or destroyed). Alternatively, the nucleus can be introduced by

injection into an oocyte and the endogenous nuclear material subsequently removed or inactivated. After such introduction, the donor cell or nucleus and oocyte are generally

fused, e.g., by electrofusion, or by use of a viral fusogen (Sendai virus) or chemical

means (e.g., use of polyethylene glycol), resulting in a nuclear transfer unit or nuclear

transfer embryo that can be cultured in vitro and implanted into a female animal to

produce cloned offspring.

Oocyte: In the present invention refers to a recipient oocyte, typically mammalian

oocyte which develops from an oogonium and following meiosis becomes a mature

ovum.

Nuclear Transfer Fusion: In the present invention, this refers to the initial product

of nuclear transfer, i.e., the product that results after insertion or infusion of a cell or

nucleus into enucleated oocyte.

Metaphase II oocyte: The preferred stage of maturation of oocytes used for

nuclear transfer (First and Prather, Differentiation, 48:1-8). At this stage, the oocyte is

sufficiently "prepared" to treat an introduced donor cell or nucleus as it does to a

fertilizing sperm.

Activation: In the present invention, this refers to the in vitro activation of a

nuclear transfer fusion by means other than via fertilization with sperm.

Cloning: In the present invention this refers to the production of an embryo or

offspring by nuclear transfer, wherein the resultant embryo or offspring has the genotype of a donor nucleus or cell, typically a somatic cell and, more preferably, a transgenic

somatic cell.

B. Detailed Description of the Invention

The present invention relates to an improved method for effecting activation of

oocytes, preferably mammalian oocytes, more preferably ungulate oocytes, and most

preferably bovine oocytes.

As discussed above, different methods for effecting parthenogenic activation of

oocytes are known. Such methods include the treatment of oocytes with ethanol,

electrical shock, cooling, calcium-free media, the use of anesthetics, electrical pulses,

calcium ionophores, IP3 and its analogues such as GTP, protein kinase inhibitors such as

puromycin and cycloheximide, and combinations thereof.(όee, Ectors et al,

Theriogenology, 44:925-933 (1995); Barnes et al, Molecular Reproduction and

Development, 36:33-41 (1993); Westhusin et al, Theriogenology, 46:243-252 (1996);

Keefer et al, Biology of Reproduction, 50:935-939 (1994); Lavoir et al, Biology of

Reproduction, 56: 194-199 (1997); and Takano H., Theriogenology, 47: 1365-1373

(1996).)

One activation protocol that has been reported to be suitable for the production of

activated bovine oocytes suitable for nuclear transfer is that of Susko-Parrish (See U.S.

Patent 5,496,720, issued March 5, 1996, and Susko-Parrish et al, Devel. Biol, 166:729- 739 (1994)). This protocol, in general, comprises activation of 10 to 52 hour bovine

oocytes by effecting the following steps in sequence:

increasing intracellular levels of divalent cation in the oocyte by introduction of

a divalent cation into the oocyte cytoplasm; and

reducing phosphorylation of cellular proteins in the oocyte wherein

phosphorylation of cellular proteins is reduced by the addition of a serine-threonine

kinase inhibitor to the oocyte in an amount effective to inhibit phosphorylation.

More specifically, the Susko-Parrish protocol comprises treating metaphase II

oocytes with ionomycin (5 μm for 4 minutes) followed by treatment with 6-

dimethylaminopurine (DMAP, 1.9 mM for 3 hours). While this method results in

successful oocyte activation (as evidenced by the development of blastocysts which give

rise to cloned embryos and offspring), this method suffers from low efficiency, i.e., the

overall percentage of treated oocytes that develop to the blastocyst stage is only about

13%. Thus, it would be beneficial if an improved oocyte activation method could be

developed that provides for higher efficiency.

Toward that end, the present inventor has developed an oocyte activation protocol

that is more efficient than that of Susko-Parrish et al (U.S. Patent 5,495,720 (1996), and

Devel Biol, 166:729-739 (1994).) This improvement is achieved by the use of

roscovitme (AG Scientific, CA) which is a specific inhibitor of cyclin dependent kinases

cdc2 and cdk2. It has been suφrisingly discovered that the use of this compound in the subject activation protocol provides for about two-fold greater efficiencies (as measured

by percentage of activated nuclear transfer fusions that give rise to blastocysts) than those

obtained according to Susko-Parrish.

Preliminary results with the inventive activation method indicate efficiencies

(percentage of oocytes that develop into blastocysts) which are significantly better than

Susko-Parrish, i.e., on the order of about 25%.

The oocytes used in the present invention may be obtained by known methods.

In general, this will comprise isolating oocytes from the ovaries of reproductive tract of

a mammal, e.g., a bovine, ovine, caprine, or porcine animal. A readily available source

of bovine oocytes is slaughterhouse materials.

Suitable mammalian sources of oocytes include primates, such as chimpanzees,

cynomolgus monkeys, sheep, pigs, cows, horses, goats, guinea pigs, mice, rats, hamsters,

etc. Most preferably, the oocytes will be obtained from bovines or other ungulates. Also,

the subject method may be used for activation of primate oocytes including human

oocytes.

For the successful use of techniques such as genetic engineering, nuclear transfer

and cloning, oocytes must generally be matured in vitro or in vivo before these cells may

be used as recipient cells for nuclear transfer, and before they can be fertilized by the

sperm cell to develop into an embryo. This process generally requires collecting

immature (prophase I) oocytes from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturing the oocytes in a maturation medium prior to

fertilization or enucleation until the oocyte attains the metaphase II stage, which in the

case of bovine oocytes generally occurs about 18-24 hours post-aspiration. For puφoses

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.

Additionally, metaphase II stage oocytes, which have been matured in vivo have

been successfully used in nuclear transfer techniques. Essentially, mature metaphase

II oocytes are collected surgically from either non-superovulated or superovulated cows

or heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic

gonadotropin (hCG) or similar hormone.

With respect thereto, the stage of maturation of the oocyte at enucleation and

nuclear transfer has been reported to affect the success of NT methods. (See e.g., Prather

et al., Differentiation's, 1-8, 1991). In general, successful mammalian embryo cloning

practices use the metaphase II stage oocyte as the recipient oocyte because at this stage

it is believed that the oocyte can be or is sufficiently"activated" to treat the introduced

nucleus as it does a fertilizing sperm. In domestic animals, and especially cattle, the

oocyte activation period generally ranges from about 10-50 hours, preferably about 28-42

hours post-aspiration. For example, immature oocytes may be washed in HEPES buffered hamster

embryo culture medium (HECM) as described in Seshagine et al., Biol. Reprod., 40, 544-

606, 1989, and then placed into drops of maturation medium consisting of 50 microliters

of tissue culture medium (TCM) 199 containing 10% fetal calf serum which contains

appropriate gonadotropins such as luteinizing hormone (LH) and follicle stimulating

hormone (FSH), and estradiol under a layer of lightweight paraffin or silicon at 39 °C.

After a fixed time maturation period, which ranges from about 10 to 40 hours, and

preferably about 16-18 hours, the oocytes will preferably be enucleated. Prior to

enucleation the oocytes will preferably be removed and placed in HECM containing 1

milligram per milliliter of hyaluronidase prior to removal of cumulus cells. This may be

effected by repeated pipetting through very fine bore pipettes or by vortexing briefly. The

stripped oocytes are then screened for polar bodies, and the selected metaphase II oocytes,

as determined by the presence of polar bodies, are then used for nuclear transfer.

Enucleation follows.

Enucleation may be effected by known methods, such as described in U.S. Patent

No. 4,994,384 which is incoφorated by reference herein. For example, metaphase II

oocytes are either placed in HECM, optionally containing 7.5 micrograms per milliliter

cytochalasin B, for immediate enucleation, or may be placed in a suitable medium, and

then enucleated later, preferably not more than 24 hours later, and more preferably 16-18

hours later. Enucleation may be accomplished microsurgically using a micropipette to remove

the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify

those of which have been successfully enucleated. This screening may be effected by

staining the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and

then viewing the oocytes under ultraviolet irradiation for less than 10 seconds. The

oocytes that have been successfully enucleated can then be placed in a suitable culture

medium.

In the present invention, the recipient oocytes will preferably be enucleated at a

time ranging from about 10 hours to about 40 hours after the initiation of in vitro

maturation, more preferably from about 16 hours to about 24 hours after initiation of in

vitro maturation, and most preferably about 16-18 hours after initiation of in vitro

maturation.

A mammalian cell nucleus of the same or different species as the enucleated

oocyte will then be transferred into the perivitelline space of the enucleated oocyte used

to produce the NT unit. The mammalian cell or nucleus and the enucleated oocyte will

be used to produce NT units according to methods known in the art. For example, the

cells may be fused by electrofusion. 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 because the membrane reforms

rapidly. Thus, if two adjacent membranes are induced to breakdown and upon reformation the 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 become one. Reference is made to U.S. Patent 4,997,384 by Prather et al.,

(incoφorated by reference in its entirety herein) for a further discussion of this process.

A variety of electrofusion media can be used including e.g., sucrose, mannitol, sorbitol

and phosphate buffered solution. Fusion can also be accomplished using Sendai virus as

a fusogenic agent (Graham, Wister lnot. Symp. Monogr., 9, 19, 1969).

Also, in some cases (e.g. with small donor nuclei) it may be preferable to inject the

nucleus directly into the oocyte rather than using electroporation fusion. Such techniques

are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994), incoφorated

by reference in its entirety herein.

Preferably, the mammalian cell or nucleus and oocyte are electrofused in a 500

μm chamber by application of an electrical pulse of 90- 120V for about 15 μsec, about 24

hours after initiation of oocyte maturation. After fusion, the resultant fused NT units are

then placed in a suitable medium until activation, e.g., HECM Heφes Culture media.

Typically activation will be effected shortly thereafter, typically less than 24 hours later,

and preferably about 4-9 hours later. The resultant nuclear transfer (NT) units will

then be activated by the improved activation protocol of the present invention.

In the case of bovine oocytes this will preferably be effected by: (i) exposing nuclear transfer units to a combination of 5 μm ionomycin, 200 μm

roscovitine (AG Scientific, CA), and 1.9 mM DMAP for about 3 ^lA to 4 1/2 minutes; and

(ii) subsequently incubating with 200 μm of roscovitine (AG Scientific, CA)

and 1.9 mM DMAP for about 3 hours.

However, it is anticipated that the amounts of these activating compounds may be

varied within wide limits. In general, it is anticipated that the amount of roscovitine may

be varied from about 10 μm to 5000 μm, more preferably 100 μm to 500 μm, the amount

of ionomycin from 0.1 to 100 μm, more preferably from 1.0 to 10.0 μm, and the amount

of DMAP from about 0.1 to 10 mM and, more preferably, from about 1 to 5 mM.

Preferably, the contact times for the first and second activation steps will

respectively range from about .1 to 10 minutes and .3 to 30 hours and, more preferably,

from about 1 to 5 minutes and 1 to 10 hours. Also, it may be possible to effect activation

in a single activation step using a combination of DMAP, ionomycin and roscovitine.

Moreover, the incubation times may be reduced with larger concentrations of activating

compounds.

While not wishing to be restricted by his belief, the present inventor believes that

roscovitine enhances development of NT embryos to the blastocyst stage because

roscovitine, unlike DMAP, is highly specific for CDK2 and CDC2. Based on this belief,

other specific inhibitors of CDK2 and CDC2 may also enhance activation efficiency and

blastocyst development, including iso-oloinoucine, olomoucine, and flavopiridol. The activated NT units may then be cultured in a suitable in vitro culture medium

until the generation of CICM cells and cell colonies. Culture media suitable for culturing

and maturation of embryos are well known in the art. For example, known media which

may be used for bovine embryo cultures and maintenance include Ham's F-l 0 + 10% fetal

calf serum (FCS), Tissue Culture Medium- 199 (TCM- 199) + 10% fetal calf serum,

Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline

(PBS), Eagle's and Whi ten's media. One of the most common media used for the

collection and maturation of oocytes is TCM- 199, and 1 to 20% serum supplement

including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer

serum. A preferred maintenance medium includes TCM- 199 with Earl salts, 10% fetal

calf serum, 0.2 mM Na pyruvate and 50 μg/ml gentamicin sulphate. Any of the above

may also involve co-culture with a variety of cell types such as granulosa cells, oviduct

cells, BRL cells, mouse embryonic feeder layers, uterine cells and STO cells.

Another maintenance medium is described in U.S. Patent 5,096,822 to Rosenkrans,

Jr. et al., which is incoφorated herein by reference. This embryo medium, named CRI,

contains the nutritional substances necessary to support an embryo. However, this

medium is not essential and many other suitable media are known and commercially

available.

In a preferred embodiment, mature oocytes will be cultured in the presence of 5

mM ionomycin, 2 mM 6-DMAP and 200 mM of roscovitine for about 3 ^lA to 4 ^lA minutes at room temperature, and then placed in ACM culture media, to which is added 2 mM of

6-DMAP at 200 mM of roscovitine for about 3 to 4 hours at 38.5 °C and 5% C0₂.

Afterward, the cultured NT unit or units will preferably be washed, e.g., with

HECM Hepes and then placed in a suitable media, e.g., ACM medium containing 10%

fetal and calf serum and contained in well plates which preferably contain a suitable

confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and

epithelial cells, e.g., fibroblasts and uterine epithelial cells derived from ungulates,

chicken fibroblasts, murine (e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell

lines, and BRL cells. Preferred feeder cells include mouse embryonic fibroblasts.

Preparation of a suitable fibroblast feeder layer is well within the skill of the ordinary

artisan.

The NT units are cultured on a feeder layer until the NT units reach a size suitable

for transferring to a recipient female, or for obtaining cells which may be used to produce

CICM cells or cell colonies. Preferably, these NT units will be cultured until at least

about 4 to 160 cells, more preferably about 4 to 128 cells, and most preferably at least

about 50 cells. The culturing will be effected under suitable conditions, i.e., about 38.5 °C

and 5% C0₂, with the culture medium changed in order to optimize growth typically

about every 2-5 days, preferably about every 3 days.

The methods for embryo transfer and recipient animal management in the present

invention are standard procedures used in the embryo transfer industry. Synchronous transfers are important for success of the present invention, i.e., the stage of the NT

embryo is in synchrony with the estrus cycle of the recipient female. This advantage and

how to maintain recipients are reviewed in Siedel, G.E., Jr. ("Critical review of embryo

transfer procedures with cattle" in Fertilization and Embryonic Development in Vitro

(1981) L. Mastroianni, Jr. and J.D. Biggers, ed., Plenum Press, New York, NY, page

323), the contents of which are hereby incoφorated by reference.

Nuclear transfer embryos produced using the subject activation protocol can also

be used to clone genetically engineered or transgenic mammals. As explained above, the

present invention is advantageous in that transgenic procedures can be simplified by

working with a differentiated cell source that can be clonally propagated. In particular,

the differentiated cells used for donor nuclei have a desired gene inserted, removed or

modified. Those genetically altered, differentiated cells are then used for nuclear

transplantation with enucleated oocytes.

Any known method for inserting, deleting or modifying a desired gene from a

mammalian cell may be used for altering the differentiated cell to be used as the nuclear

donor. These procedures may remove all or part of a gene, and the gene may be

heterologous. Included is the technique of homologous recombination, which allows the

insertion, deletion or modification of a gene or genes at a specific site or sites in the cell

genome. Activated nuclear transfer fusions produced according to the present invention can

be used to provide adult mammals with desired genotypes. Multiplication of adult

ungulates with proven genetic superiority or other desirable traits is particularly useful,

including transgenic or genetically engineered animals, and chimeric animals.

Furthermore, cell and tissues from the NT fetus, including transgenic and/or chimeric

fetuses, can be used in cell, tissue and organ transplantation for the treatment of numerous

diseases as described below in connection with the use of CICM cells.

For production of CICM cells and cell lines, after NT units of the desired size are

obtained, the cells are mechanically removed from the zona pellucida, and are then used.

This is preferably effected by taking the clump of cells which comprise the NT unit,

which typically will contain at least about 50 cells, washing such cells, and plating the

cells onto a feeder layer, e.g., irradiated fibroblast cells. Typically, the cells used to

obtain the stem cells or cell colonies will be obtained from the inner most portion of the

cultured NT unit which is preferably at least 50 cells in size. However, NT units of

smaller or greater cell numbers, as well as cells from other portions of the NT unit, may

also be used to obtain ES cells and cell colonies. The cells are maintained in the feeder

layer in a suitable growth medium, e.g., alpha MEM supplemented with 10% FCS and

0.1 mM β-mercaptoethanol (Sigma) and L- itamine. The growth medium is changed

as often as necessary to optimize growth, e.g., about every 2-3 days. This culturing

process results in the formation of CICM cells or cell lines. One skilled in the art can vary the culturing conditions as desired to optimize growth of the particular CICM cells.

Also, genetically engineered or transgenic mammalian CICM cells may be produced

according to the present invention. That is, the methods described above can be used to

produce NT units in which a desired gene or genes have been introduced, or from which

all or part of an endogenous gene or genes have been removed or modified. Those

genetically engineered or transgenic NT units can then be used to produce genetically

engineered or transgenic CICM cells, including human cells.

The resultant CICM cells and cell lines, preferably human CICM cells and cell

lines, have numerous therapeutic and diagnostic applications. Most especially, such

CICM cells may be used for cell transplantation therapies. Human CICM cells have

application in the treatment of numerous disease conditions. Human NT units er se may

also be used in the treatment of disease conditions.

In this regard, it is known that mouse embryonic stem (ES) cells are capable of

differentiating into almost any cell type, e.g., hematopoietic stem cells. Therefore, human

CICM cells produced according to the invention should possess similar differentiation

capacity. The CICM cells according to the invention will be induced to differentiate to

obtain the desired cell types according to known methods. For example, the subject

human CICM cells may be induced to differentiate into hematopoietic stem cells, muscle

cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells,

etc., by culturing such cells in differentiation medium and under conditions which provide for cell differentiation. Medium and methods which result in the differentiation of CICM

cells are known in the art as are suitable culturing conditions.

For example, Palacios et al, Proc. Natl. Acad. Set, USA, 92:7530-7537 (1995)

teaches the production of hematopoietic stem cells from an embryonic cell line by

subjecting stem cells to an induction procedure comprising initially culturing aggregates

of such cells in a suspension culture medium lacking retinoic acid followed by culturing

in the same medium containing retinoic acid, followed by transferral of cell aggregates

to a substrate which provides for cell attachment.

Moreover, Pedersen, J. Reprod. Fertil Dev., 6:543-552 (1994) is a review article

which references numerous articles disclosing methods for in vitro differentiation of

embryonic stem cells to produce various differentiated cell types including hematopoietic

cells, muscle, cardiac muscle, nerve cells, among others.

Further, Bain et al, Dev. Biol, 168:342-357 (1995) teaches in vitro differentiation

of embryonic stem cells to produce neural cells which possess neuronal properties. These

references are exemplary of reported methods for obtaining differentiated cells from

embryonic or stem cells. These references and in particular the disclosures therein

relating to methods for differentiating embryonic stem cells are incoφorated by reference

in their entiretv herein.

Thus, using known methods and culture medium, one skilled in the art may culture

the subject CICM cells, including genetically engineered or transgenic CICM cells, to obtain desired differentiated cell types, e.g., neural cells, muscle cells, hematopoietic

cells, etc. The use of CICM cells for cell therapies is disclosed in U.S. Serial

Nos.08/781,752 and 09/004,606, filed January 10, 1997 and January 8, 1998,

respectively, the contents of which are incoφorated by reference in their entirety herein.

In order to more clearly describe the subject invention, the following examples are

provided.

EXAMPLES

Oocyte collection. Ovaries were obtained from slaughter adult cows, placed in

30 °C saline solution and transported to the laboratory. Oocytes were aspirated from

ovarian follicles and evaluated for the presence of cumulus cells.

Oocyte maturation. Maturation was performed in TCM 199 media plus FSH and

LH at 38.5 °C and 5% CO₂ for 24 hours.

Oocyte activation. Twenty-four hours post maturation, oocytes were placed in

HECM Hepes culture media.

With the addition of 5 microM of inomomycin, 2 mM or 6-DMAP and 200

microM or roscovitine for three and a half to four and a half minutes at room

temperature and then placed in ACM culture media.

To the above medium is added 2 mM of 6-DMAP and 200 microM of roscovitine

and the oocytes are cultured for three to four hours at 38.5° C and 5% CO₂. After this

incubation, oocytes were rinsed four times in HECM Hepes and placed in culture in ACM

media plus 10% fetal calf serum for seven and a half days using mouse embryonic feeder

layer as a co-culture. Media was changed at day four of culture. After seven and a half

days, blastocysts were counted, fixed with paraformaldehyde, and stained with HOECHST. Total cell number was counted using UV light and only embryos with more

than 50 cells were considered blastocysts.

Claims

WHAT IS CLAIMED IS:

1. An improved method for activation of mammalian oocytes for use in

nuclear transfer that comprises (i) contacting said oocytes with a source of divalent

calcium, and (ii) contacting said oocytes with a serine threonine kinase inhibitor, wherein

said steps are effected in sequence or in combination, wherein the improvement

comprises additionally contacting said oocytes with a CDC2 or CDK2 inhibitor selected

from the group consisting of roscovitine, iso-olomoucine, olomoucine, and flavopiridol.

2. The method of Claim 1 , wherein the source of divalent cation is ionomycin

and the serine threonine kinase inhibitor is 6-dimethylaminopurine (DMAP).

3. The method of Claim 1, wherein steps (i) and (ii) are effected in sequence

and roscovitine is used in both step (i) and (ii).

4. The method of Claim 3, wherein the amount of roscovitine ranges from

about 20 μm to 2000 μm.

The method of Claim 4, wherein the amount of roscovitine is about 200 μm.

6. The method of Claim 1 , wherein step (i) comprises contacting said oocytes

with a mixture of ionomycin, roscovitine and DMAP and step (ii) comprises contacting

said oocytes with a mixture of roscovitine and DMAP.

7. The method of Claim 6, wherein step (i) is effected for about 3 A to 4 A

minutes using 200 μm roscovitine, 1.9 mM DMAP and 5 μm ionomycin, and step (ii) is

effected by contacting oocytes for about 3 hours with 200 μm roscovitine and 1.9 mM

DMAP.

8. The method of Claim 1, wherein the mammalian oocytes are selected from

the group consisting of non-human primate oocytes, mouse oocytes, human oocytes, rat

oocytes, mouse oocytes, bovine oocytes, ovine oocytes, porcine oocytes, hamster oocytes,

caprine oocytes, and guinea pig oocytes.

9. The method of Claim 1, wherein the mammalian oocytes are bovine

oocytes.

10. The method of Claim 1 , wherein the activated oocytes are cultured to

produce a blastocyst.

11. The method of Claim 10, wherein said oocytes are cultured to produce an

embryo suitable for implantation into a female recipient.

12. The method of Claim 1 , wherein the oocytes comprise mammalian oocytes

which have been enucleated and a donor cell or nucleus inserted therein.

13. The method of Claim 12, wherein said mammalian oocytes comprise

ungulate oocytes and the donor cell or nucleus is of ungulate origin.

14. The method of Claim 13, wherein said donor cell or nucleus is a somatic

cell.

15. The method of Claim 14, wherein said somatic cell or nucleus is obtained

from an actively dividing mammalian cell culture.

16. The method of Claim 14, wherein said somatic cell is selected from the

group consisting of fibroblasts, keratinocytes-, intestine, stomach, cumulus, lung, heart,

skin, reproductive organ cell, esophagus lymphocyte, macrophage, kidney, and neural

cells.

17. The method of Claim 15, wherein said somatic cell has been genetically

modified by addition, substitution, or deletion of a DNA sequence.

18. The method of Claim 17, wherein said genetically modified somatic cell is

a fibroblast.

19. The method of Claim 17, wherein said genetically modified cell comprises

an introduced human gene.

20. The method of Claim 19, wherein said human gene is selected from the

group consisting of an albumin, collagen, growth factor, cytokine, hormone, enzyme or

an antibody gene.