Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

• the present invention is generally directed to a process for multiplying (cloning) embryos and specifically directed to a process for producing large numbers of identical mammalian embryos by introducing embryonic stem cell nuclei into enucleated recipient oocytes, enucleated two cell embryos, or replacing the inner cell mass of a mammalian blastocyst with donor mammalian embryonic stem cells. Accordingly, the present invention is also generally directed to a process for cloning live animals by introducing embryonic stem cell nuclei into enucleated recipient oocytes, enucleated two cell embryos, or replacing the inner cell mass of a mammalian blastocyst with donor mammalian embryonic stem cells.
• Embryonic stem cells are pluripotent cells directly derived from the inner cell mass of blastocysts (Evans, M.J. et al. , Nature 292: 154-156 (1981);
• Embryonic stem cells are able to form permanent cell lines in vitro.
• Mouse embryonic stem cells can be cultured over at least sixty passages and retain, in a majority of cases, a normal karyotype.
• Embryonic stem cells have been shown to remain in undifferentiated form in vitro if maintained on embryonic fibroblast feeder cell layers. In cell suspension, they will begin differentiation, containing elements of glandular, heart, skeletal smooth muscle, nerve, keratin-producing cells, and melanocytes (Doetschman, T.C. et al, Dev. Biol. 127:224-221 (1988)).
• Embryonic stem cells are the most pluripotent cultured animal cells known. When embryonic stem cells are injected into an intact blastocyst cavity, or under the zona pellucida, at the morula stage embryo, they are capable of contributing to all somatic tissues, including the germ line, in the resulting chimeras (rev'd by Bradley, A., Curr. Op. Cell. Biol 2: 1013-1017
• WO 90/03432 discloses pluripotential embryonic stem cell-like cells derived from porcine and bovine species. This international application describes the production of pluripotential ungulate embryonic stem cells, together with details of the morphology enabling recognition of the cells. However, there is no confirmed evidence that any of these cells are true embryonic stem cells.
• Embryonic stem cells can be cultured and manipulated in vitro and then returned to the embryonic environment to contribute to all tissues including the germ line (for a review, see Robertson, E.J., Trends in Genetics 2:9-13 (1986); Evans, M.J., Mol. Bio. Med. 6:551-565 (1989); Johnson, M.P. et al , Fetal Ther. 4 (Suppl. l):28-39 (1989); Babinet, C. et al, Genome 57:938-949 (1989)).
• embryonic stem cells propagated in vitro contribute efficiently to the formation of chimeras, including germ line chimeras, but in addition, these cells can be manipulated in vitro without losing their capacity to generate germ line chimeras (Robertson, E. J. et al. , Nature 525:445-447 (1986)).
• Embryonic stem cells in vitro can be modified by any of the techniques currently known for carrying out manipulation of genetic material.
• the current methods for gene transfer into cells include transfection, cell fusion, electroporation, microinjection, DNA viruses, and RNA viruses.
• Embryonic stem cells in vitro can be modified by any of these techniques and then introduced back into the embryonic environment for expression and subsequent transmission to progeny animals. Cloning of Identical Mammals
• Blastocysts derived from half embryos are smaller than normal blastocysts, but they develop into normal conceptuses.
• Embryonic stem cells have been used to study in vivo events in embryo chimeras. The most commonly used method is the injection of several embryonic stem cells into the blastocoel cavity of intact blastocysts (Bradley, A. et al, Nature 509:255-256 (1984)).
• An alternative method for germ line chimera production involves sandwiching a clump of embryonic stem cells between two eight-cell embryos (Bradley, A. et al, in Teratocarcinomas and
• Embryomc Stem Cells A Practical Approach, ed. Robertson, E.J. (IRL, Oxford, U.K.), pp. 113-151 (1987); Nagy, A. et al , Development 770:815- 821 (1990)). Both methods result in germ line transmission at high frequency.
• an alternative method for the production of embryonic stem cell- embryo chimeras with high levels of chimerism in most, if not all tissues, including the germ line involves the co-culture of embryonic stem cells with eight-cell embryos, and follows the same principles as those described for the aggregation of pre-implantation embryos (Tarkowski, A.K., Nature 790:857 (1961); Mintz, B., J. Exp.
• the present invention provides a process wherein embryonic stem cells are used as cellular or nuclear donors to produce large or unlimited numbers of genetically identical animals and/or embryos, especially of mammalian origin.
• embryonic stem cell nuclei are introduced into enucleated oocytes.
• embryonic stem cell nuclei are introduced into blastomeres of enucleated two celled embryos.
• whole embryonic stem cells are introduced into a recipient blastocyst from which the inner cell mass has been removed.
• FIG. 1 Fusion of cytoplasts carrying embryonic stem cell nuclei with enucleated oocytes and two celled embryos ("indirect fusion")
• Unfertilized zona-free oocytes are divided mechanically into several fragments, or small cytoplasts are removed by micropipette from zona-intact oocytes. Each cytoplast is brought into contact with two to five embryonic stem cells. Such aggregates are then subjected to fusion with PEG, and cytoplasts that have fused with a single embryonic stem cell are injected into enucleated oocytes and two celled embryos. Following microinjection, the oocytes or two celled embryos are exposed to an electric field.
• FIG. 1A Methods of preparation of inner cell mass-free blastocysts
• a small opening in the zona pellucida (above the inner cell mass) is made either by partial zona dissection using a fine glass needle, or by treatment by acidified Tyrode's solution (step 1).
• the manipulated blastocysts are cultured in M16 medium at 37 °C for six to 12 hours (step 2).
• the cultured blastocysts undergo expansion, and a small vesticle, usually containing the whole inner cell mass, is extruded through the opening in the zona.
• the extruded inner cell mass is cut off by a glass needle or surgical blade (step 3).
• FIG. 2B Methods of preparation of inner cell mass-free blastocysts
• Step 1 The zona pellucida and polor trophectoderm (at the top of the inner cell mass) is punctured by an injection pipette.
• Step 2 A fragment of the inner cell mass is sucked into the pipette.
• Step 3 The blastocyst is released from the holding pipette, turned over, and sucked again into the holding pipette exactly where the inner cell mass fragment was previously withdrawn.
• Step 4 By increasing the negative pressure in the holding pipette, the inner cell mass is subsequently removed from within the blastocyst and cut off by a glass needle or surgical blade.
• Step 5 The inner cell mass-free blastocysts are ready for injection (if they have not collapsed) or are cultured for a couple of hours in order to re- expand (if collapsed).
• embryonic stem cells provide an unlimited source of theoretically identical cells. Their pluripotency, their ability to respond and to follow normal developmental signals, and their uniform genetic makeup, confer on these cells tremendous value for cloning by nuclear transfer or other techniques.
• Embryonic stem cells thus provide a route for the generation of transgenic animals, a route which has a number of important advantages compared with more conventional techniques, such as zygote injection and viral infection (Wagner, T.J. et al. , in Experimental Approaches to Embryonic Development, J. Rossant, et al , eds., Cambridge University Press (1986)), for introducing new genetic material into such animals.
• the gene of interest can be introduced and its integration and expression characterized in vitro.
• the effect of the introduced gene on the embryonic stem cell growth can be studied in vitro.
• the characterized embryonic stem cells having a novel introduced gene can be efficiently introduced into embryos by blastocyst injection or embryo aggregation and the consequences of the introduced gene on the development of the resulting transgenic chimeras monitored during pre- or post-natal life.
• the site in the embryonic stem cell genome at which the introduced gene integrates can be manipulated, leaving the way open for gene targeting and gene replacement (Thomas, K.R. et al, Cell 57:503-512 (1987)).
• the present invention is based on a process designed by the inventors wherein cloned animals are produced from embryonic stem cells.
• the inventors have discovered that when embryonic stem cell nuclei are introduced into enucleated oocytes or two celled embryos, or whole embryonic stem cells introduced into blastocysts from which the inner cell mass has been removed, they can promote fetal development.
• the inventors have found a process to utilize all of the advantages and desirable characteristics of embryonic stem cells, discussed above, for the production of cloned animals.
• embryonic stem cells can be created from any genetically superior animal or from an animal with specific desirable genetic traits, and passaged in vitro to produce theoretically unlimited numbers of cells as a donor source for the formation of cloned animals.
• These cloned animals may have the same genetic makeup as the donor embryonic stem cell or may contain a genome which has been developed by manipulation of the embryonic stem cell prior to cloning and subsequent development.
• one embodiment of the invention is directed to a method of producing cloned animals comprising introducing an embryonic stem cell from any desired animal of any animal species into a blastocyst of said animal species, from which blastocyst the inner cell mass has been removed.
• the stem cell may or may not be transgenic.
• the blastocysts may be collected by methods known in the art following mating of the desired animals.
• the inner cell mass is removed by creating a small opening in the zona pellucida, allowing the blastocyst to expand, allowing the extrusion, through the opening, of a small vesicle, usually containing the whole inner cell mass, and the removal of the extruded inner cell mass from the blastocyst.
• the zona pellucida and polar trophectoderm are punctured by an injection pipette, and a fragment of the inner cell mass is pulled outside the zona pellucida by suction into the pipette.
• the blastocysts should not be too small (i.e., they should have a well developed cavity), not collapsed, and still within the zona. In the method described in Figure 2A, the whole polar trophectoderm is also removed.
• mouse embryos are removed at the four to eight cell stage, put in M16 medium, and cultured at 40°C for about three days.
• the result is a trophoblastic vesicle devoid of any inner cell membrane because the high temperature prevents the inner cell membrane from forming.
• the high temperature treatment can be applied to other animals at the comparable developmental stage, although the exact temperature may not be 40°C.
• the optimal temperature can be determined by empirical observation.
• the injection of embryonic stem cells into blastocysts may be accomplished by methods well-known in the art. For example, for the present invention, embryonic stem cells are introduced into blastocysts by the method of Bradley (in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (ed.
• the inner cell mass is removed from blastocysts when the inner cell mass is clearly defined, i.e., forms a discrete mass.
• more than one embryonic stem cell is introduced into the blastocyst.
• the inner cell mass contains around 10-15 cells.
• ten cells are injected into the mouse blastocyst.
• embryonic stem cell or “embryonic stem cell” is intended those cells that retain the developmental potential to differentiate into all somatic and germ cell lineages.
• the embryonic stem cells or pluripotential cells of the present invention include, but are not limited to, cells derived from humans, mice, sheep, pigs, cattle, and goats.
• a method for producing cloned animals comprising introducing an embryonic stem cell nucleus from an animal species into an oocyte of said animal species, from which oocyte the nucleus has been removed. Oocytes are flushed from the oviducts of females that have been induced to ovulate or spontaneously ovulate according to methods well-known in the art.
• the embryonic stem cell nucleus may be introduced into the oocyte by methods standard in the art. For example, see Czolowska, R. et al. , J. Cell.
• nuclear transfer is accomplished by indirect fusion involving polyethylene glycol (PEG) fusion of embryonic stem cells and oocyte fragments or cytoplasts and subsequent fusion of single embryonic stem cells and an oocyte in an electric field.
• PEG polyethylene glycol
• Unfertilized oocytes are divided mechanically into several fragments (zona-free oocytes) or small cytoplasts are formed from zona-intact oocytes. The cytoplasts or fragments are then exposed to embryonic stem cells to form aggregates. Aggregates are then treated for PEG fusion according to methods known in the art. For example, see Czolowska, R. et al. , J. Cell Sci. 69:19-34 (1984). Cytoplasts or fragments fused with a single embryonic stem cell are then injected under the zona pellucida of oocytes. Following injection, the oocytes are exposed to an electric field according to methods known in the art.
• nuclear introduction is by direct fusion in an electric field.
• oocytes with a single embryonic stem cell introduced under the zona pellucida are exposed to an electric field treatment (electrofusion) according to methods known in the art.
• Fused oocytes containing the embryonic stem cell nuclei are then activated prior to implantation into a pseudopregnant female.
• activation is with 8% ethanol. The method of ethanol activation is described by Cuthbertson, K.S.R., J. Exp. Zool 226:331-314 (1983).
• oocyte for the purposes of the present invention, as used herein for the recipient cell, is intended a cell which develops from an oogonium and, following meiosis, becomes a mature ovum. Not all oocytes are equally optimal cells for efficient donation of an embryonic stem cell in the mouse. For purposes of the present invention, mid metaphase II stage oocytes have been found to be optimal. Mature metaphase II oocytes may be collected surgically from either non-superovulated or superovulated mammals at a predetermined time past the onset of estrus or past an injection of human chorionic gonadotropin (hCG) or similar hormone. Methods of superovulation in domestic animals are routine in the art. Alternatively, immature oocytes may be removed by aspiration from ovarian follicles obtained from slaughtered animals and then may be matured in vitro by appropriate hormonal treatment and culturing.
• hCG human chorionic gonadotropin
• a method for producing cloned animals comprising introducing an embryonic stem cell nucleus from an animal species into a two-celled embryo of said animal species, from which two-celled embryo the nuclei of both blastomeres have been removed.
• Embryos at the two-celled stage are collected from females by flushing the oviduct following ovulation or superovulation (which may be by hCG injection).
• embryonic stem cells are introduced into one of the blastomeres of the two-celled embryo under the zona pellucida.
• cytoplasts or fragments of oocytes that have been fused with a single embryonic stem cell as described above are injected under the zona pellucida. Following microinjection, the embryo is exposed to an electric field as described above for "indirect fusion. " For in vivo development, embryos are then transplanted into the oviducts of pseudopregnant females.
• two-celled embryo is intended an embryo after the first post-fertilization cleavage division in which the daughter cells have both completed mitosis, have separate nuclei with nuclear membranes, and have complete individual outer plasma membranes.
• embryonic stem cells at low passage number are introduced into the blastocyst, or provide nuclei for transfer into the embryo or oocyte.
• the inner cell mass (10-15 cells in mouse) from a blastocyst is introduced into primary culture. After cell divisions leading to the formation of several million cells, the cells are then frozen and stored according to standard procedures (e.g., 7.5 % DMSO, packed in styrofoam, allowed to freeze in a mechanical freezer at a rate of around l°C/minute, and then transferred to liquid nitrogen storage). Aliquots of the frozen cells are then used to re-seed a fresh culture, and cells grown in this first passage culture are then used as the donors for the blastocysts.
• standard procedures e.g., 7.5 % DMSO, packed in styrofoam, allowed to freeze in a mechanical freezer at a rate of around l°C/minute, and then transferred to liquid nitrogen storage. Aliquots of the frozen cells are then used to
• the cytoplasts are exposed to two to five stem cells. Since cells have a tendency to disaggregate from the cytoplast, initial incubation with several cells is preferred.
• the oocytes or two-cell embryos are placed in hypotonic medium (50% M2) for around 15 minutes prior to electrofusion.
• hypotonic medium 50% M2
• the volume of oocyte/two-cell embryo is diminished, and the perivitelline space becomes larger.
• the donor embryonic stem cell floating freely in the perivitelline space, does not have contact with the egg cell membrane. Therefore, areas of contact are preferably expanded as herein described. After fusion (of the stem cell plasma membrane and recipient cell plasma membrane), the donor nucleus is released into the recipient cytoplasm.
• a double pulse is used for fusing embryonic stem cells with oocytes or 2-cell embryos. Two 60 V pulses of 30 ⁇ seconds apart are administered. Immediately after fusion, the embryos/oocytes are transferred to 37 °C.
• a 0.3 M glucose solution is used as the electrofusion buffer.
• the embryonic stem cells are genetically engineered prior to introduction into the blastocyst, oocyte or embryo. Genetic manipulation can be accomplished by any of the procedures known in the art; for example, by recombinant retrovirus infection, which retrovirus contains genetic material which is desired to introduce into the embryonic stem cell, by transfection procedures, by electroporation procedures, by microinjection, and the like.
• retrovirus infection which retrovirus contains genetic material which is desired to introduce into the embryonic stem cell
• transfection procedures by electroporation procedures, by microinjection, and the like.
• Examples of desirable genes to be introduced into the embryonic stem cells include, but are not limited to, the genes that control the number of ovulations, litter size, seasonality, prenatal survival, and the determination of sex.
• animal any living creature that contains cells in which the methods of the present invention can be practiced. Foremost among such animals are animals valuable for the livestock industry; however, the invention is not intended to be so limiting, it being within the contemplation of the present invention to apply the methods of the present invention to any and all animals for which clones are desirable.
• Embryonic stem cells were prepared essentially by the method of Robertson described in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (ed. E.J. Robertson) IRL Press, Oxford, Wasington, D.C. (1987), Chapter 4, "Embryo-Derived Stem Cells, specifically pp. 71-112," except that the feeder layer is derived from C578L/6J 12-14 day fetuses, and delayed blastocysts were not used as a source of starting materials.
• Inner cell masses were obtained from early four-day blastocysts originating from spontaneously ovulating Fl (C57/BL x CBA/H or C3H x C57/BL) female mice mated either with C57/BL or Fl (C57/BL x CBA/H) males. Isolation of the inner cell mass was performed by treatment with A 23187 calcium ionophore (Sigma) according to the method described by Surani et al. (J. Embryol Exp. Morph. 45:237-247 (1978)).
• the trophectoderm-free inner cell masses were either directly used for experiments or were placed into Ca ++ and Mg ++ free M2 medium (Sigma) for 30-40 minutes and then dissociated into two to three fragments. Prior to microinjection both whole inner cell masses and inner cell mass fragments were incubated in M2 medium containing 1 ⁇ g/ml of Cytochalasin D (CD).
• CD Cytochalasin D
• Recipient blastocysts were obtained from females of randomly bred Swiss Albino mice, from the inbred BALB/cByJ strain and from the albino standard Fl hybrid strain CAF1/J. In the majority of experiments, spontaneously ovulated females mated with Swiss albino or BALB/cByJ males were used. Blastocysts were collected 3.5 day after mating. After flushing, blastocysts were placed in M2 medium and the inner cell mass was removed using two different methods (Fig. 2A and 2B). In the first method (Fig. 2A and 2B). In the first method ( Figure
• a small opening in the zona was made either by treatment with acidified Tyrode's solution (zona drilling) or by partial zona dissection similar to that described by Malter and Cohen (Gamete Res. 24:61 '-80 (1989)).
• Blastocysts were placed in M16 medium (Sigma) (Whittingham, D.G., J. Reprod. Fertil. (Suppl) 74:7-21 (1971)) in 5% CO 2 in air at 37°C.
• the expanding blastocysts extruded a small vesicle through the opening in the zona, usually containing the whole inner cell mass.
• the extruded inner cell mass was cut off by a glass needle or surgical blade (No.
• the blastocysts did not collapse and were ready for immediate injection. Those which collapsed were cultured in M16 medium until they re- expanded. In some cases, the blastocysts with the extruded inner cell mass were injected with embryonic stem cells prior to removing the inner cell mass.
• the zona pellucida and polar trophectoderm at the top of the inner cell mass, were punctured by an injection pipette and a fragment of the inner cell mass was sucked gently into the pipette and pulled outside the zona pellucida.
• the blastocyst was released from the holding pipette, turned over and sucked into the holding pipette exactly where the inner cell mass fragment was previously withdrawn.
• the inner cell mass was subsequently removed from the blastocyst and cut off as described previously.
• the blastocysts were kept in M2 medium for 1-2 hours in order to allow them to re-expand and then were transferred into oviducts of pseudopregnant CD1 or Swiss Albino females mated with vasectomized Swiss albino or BALB/cByJ males, at the first day of pregnancy (day of the vaginal plug). Starting from day 9, a composition of the vaginal smear was checked twice a day. When erythrocytes appeared in a smear, this suggests the beginning of resorption of an embryo. Accordingly, at this time, the recipient female was killed and the embryos removed. After morphological examination the embryos were processed for histology.
• Non-injected inner cell mass-free blastocysts were transferred into pseudopregnant recipients to test if the method of inner cell mass removal ensured the elimination of embryonic material.
• a single young was born and a second one obtained after a Caesarian section at day 19.
• the first new-born young derived from E9 embryonic stem cells, was small, had a large subcutaneous hemorrhage in the posterior part of the body and died after few hours.
• the second one obtained from D3 embryonic stem cells, was transferred to a foster mother. It grew very slowly and even with intensive care, died at day 10 after birth.
• blastocelic fluid Pulmonary et al, Proc. Natl Acad. Sci.
• a small number of cells (at least up to 3) is not able to form a functional inner cell mass (Snow, M.H.L., J. Embryol Exp. Morph. 55:81-86 (1976); Markert, C.L. et al, Science, 202:56-58 (1978)).
• One-third of the inner cell mass (around 3-5 cells) cannot sustain development of an embryo (these experiments). Since full term development was obtained (in these studies) after transfer of both whole and half of the inner cell mass into inner cell mass-free blastocysts, however, this shows that this method of blastocyst reconstruction is effective.
• embryonic stem cells originated from different mouse strains (D3 - 129/SvJ and E9 - C57B1/6J). This shows that development to term is also independent of the strain of origin of embryonic stem cell lines. Since in both embryonic stem lines used in the experiments herein, normal diploid cells constituted over 90% of cell population, the detrimental effects of aneuploidy can be excluded, as can the influence of mutation accumulated in a specific cell line. Thus, it might be that during prenatal development, due to a certain incompatibility between the two fetal components of different origin (trophectodermal derivatives and embryonic stem cell derived embryonic tissues), the growing conceptus is nutritionally affected.
• D3 available from the American Tissue Culture Collection, is a male (XY) line from the 129/SvJ strain of mouse.
• E9 was derived by the inventors from the C57BL/6J strain using the modification of Robertson described in Example 1 herein.
• the embryonic stem cells were cultured either on mitomycin C treated fibroblast cells (from day 12 C57BL/6J mouse fetuses) or in the absence of feeder layer by supplementation with conditioned media containing leukaemia inhibitory factor (Smith, A.G. et al, Nature 336: 688-690 (1988); Smith, A.G. et al, J. Reprod. Fert. 84:619-624 (1988) Williams, R.L. et al, Nature
• Embryonic stem cells were grown in 100 mm cell culture Petri dishes pretreated with a 0.1 % porcine gelatine solution for at least 1 hour.
• the medium for the culture of embryonic stem cells was composed of alpha-MEM supplemented with 20% FCS (CC Biologicals, Cleveland, OH, USA), 0.1 mM
• embryonic stem cells were trypsinized (Robertson, E. et al, Nature 525:445-448 (1987)), placed in an equal volume of medium to inactivate the trypsin, and centrifuged at 1500 rpm (Sorvall RT).
• mice of the strain Balb/cByJ were used as donors of oocytes and cleaving eggs. Females were kept in a 12 hour day/night regime and induced to ovulate by injection of 5 i.u. PMSG (Sigma) and 5 i.u. hCG (Sigma) given 44-48 hours apart. Oocytes were flushed from the oviducts either 14-14.5 hours (early oocytes) or 16.5-17.5 hours (late oocytes) after hCG injection and treated with 1 mg/ml hyaluronidase (Gibco) to remove the cumulus cells. To obtain 2-cell embryos, the hormonally treated females were mated to Balb/cByJ males. The early, mid and late 2-cell stage embryos were collected 34-44 and 52-53 hours after hCG injection, respectively.
• oocytes, 2-cell embryos and embryonic stem cells Prior to micromanipulation, all oocytes, 2-cell embryos and embryonic stem cells were preincubated for at least 20 min in CD containing M2 medium (1 /-ig/ml) at room temperature.
• embryonic stem cells were introduced (through the same opening in the zona pellucida) into the perivitelline space.
• one (or sometimes two) embryonic stem cell was introduced whereas 3-5 cells were transferred under the zona pellucida of 2-cell embryos.
• 3-5 cells were transferred under the zona pellucida of 2-cell embryos.
• two-cell embryos are less likely to fuse as frequently with embryonic stem cells as oocytes are. Therefore, in preferred embodiments, it is necessary to increase the fusion efficiency by introducing a higher number of cells.
• the embryonic stem cells were also introduced under the zona pellucida of non-enucleated oocytes and 2-cell embryos and they were treated by electric field only ("Direct fusion" - see below).
• the cytoplasts were then returned to the well containing embryonic stem cells. Each cytoplast was brought into contact with 2-5 embryonic stem cells. Aggregates were treated for PEG (Fluka, mol.w. 1500) fusion as described by Czolowska, R. et al, J. Cell Sci., 69:19-34 (1984)). Only cytoplasts which fused with a single embryonic stem cell were selected. These were incubated in CD-containing M2 medium and injected under the zona pellucida of oocytes and 2-cell embryos. Following microinjection, the oocytes/2-cell embryos were exposed to an electric field (see below).
• Oocytes and 2-cell embryos containing embryonic stem cells introduced under the zona pellucida were incubated for 30 min in 50% M2 plus CD medium at 37 °C in order to enable close contact between the cells to be fused. Before exposure to electric field treatment, they were washed in the same type of solution which was subsequently used for electrofusion.
• Oocytes/2-cell embryos carrying embryonic stem cells were placed in the electrofusion chamber filled with glucose, between two parallel platinum electrodes located 0.3 mm apart with the contact plane perpendicular to the direction of electric field vector (Kubiak, J.Z. et al, Exp. Cell Res. 757:561- 566 (1985); Ozil, J.P. et al, J. Embryol. Exp. Morph., 96:211-228 (1986)).
• Different fusion parameters were tested in order to establish the best fusion efficiency and the lowest frequency of lysis of fused cells. Two types of treatment were chosen for further experiments:
• the oocytes/2-cell embryos were placed in preheated 37°C M2 plus CD medium and checked under Nomarski's optics every 10 minutes. Fusions usually occurred within 5-20 minutes. Occasionally, the non-fused aggregates were treated once again using the same fusion parameters.
• Oocytes from the experimental and control groups were activated by exposure to a solution of 8% ethyl alcohol in M2 medium for 5 minutes at
• activated oocytes were cultured (prior to transfer to M16 - CD containing medium) for 5-6 hours. This treatment suppresses the extrusion of the second polar body and results in the formation of diploid oocytes.
• the developmental abilities of activated oocytes were tested after 3-4 days of in vivo culture in the oviducts of immature females (approx. 3 weeks old).
• oocytes and 2-cell embryos were cultured in M16 medium at 37°C in 5% CO 2 in air.
• oocytes/2 cell - embryos were transplanted either to the oviducts of pseudopregnant CDl recipients mated with vasectomized Balb/cByJ males (for pre- and postimplantation development) or into ligated oviducts of immature (3-4 week old) CDl females (for preimplantation development only).
• the obtained preimplantation embryos derived from reconstituted oocytes and 2-cell embryos were fixed in Heidenhein's fixative, whole- mounted (Tarkowski, A.K. et al, J. Embryol. Exp. Morph. 78: 155-180 (1976)) and stained with Harris hematoxylin.
• Oocytes fuse more easily with embryonic stem cells than do 2-cell embryos (40%-90% vs. 15%-35% - Table 1.). Also, early oocytes and embryos fuse better than the late ones. Differences in the fusogenic abilities between oocytes and 2-cell embryos were even more significant since the majority of oocytes was able to fuse when a single embryonic stem cell only was introduced into the perivitelline space, whereas in order to obtain fusions between 2-cell embryos and a single embryonic stem cell, 3-5 cells had to be introduced under the zona pellucida (see Materials and Methods).
• Embryonic stem cell nuclei underwent premature chromosome condensation (PCC) when introduced to the cytoplasm either of intact or enucleated oocytes.
• PCC premature chromosome condensation
• the dispersion of the nuclear envelope and a disappearance of nucleoli were observed 15-30 minutes after fusion. Although no direct comparison was carried out, it appears that PCC often took place earlier in intact oocytes than in enucleated ones.
• PCC was completed and the condensed chromatin was arranged in more or less regular PCC figures.
• the number of reconstituted early oocytes that were activated after ethanol treatment was relatively high (approx. 70-80% of the treated oocytes in some experiments), and similar to that observed in the control group.
• early oocytes usually 16.5-17.5 hours after hCG (at the fusion-activation time)
• the nuclei grew rapidly during the first 5-6 hours, and reached at least the size of full grown pronuclei at the end of the first cell cycle.
• embryonic stem cell nuclei behave normally by swelling (increasing in size) and stain as do nuclei containing decondensed chromatin.
• the nuclei may remain in a condensed state (darkly staining).
• the introduced embryonic stem cell nuclei swelled enormously reaching, in the most extreme cases, three-fourths of the diameter of an oocyte (i.e. 40% of an oocyte's total volume).
• activated oocytes receiving embryonic stem cell nuclei extruded "a second polar body" 1.5 hours after activation, i.e., at the normal time for the fertilized or artificially activated oocytes, if they were cultured in CD free M16 medium.
• nuclei In late oocytes, nuclei also swelled rapidly but only occasionally fo ⁇ ned the "gigantic" nuclei observed in early oocytes. Extrusion of "a second polar body” sometimes also occurred. However, fragmentation more frequently was observed in aged, activated oocytes (if they are cultured in CD free M16 medium). Since fragmentation, or rather increased cortical activity leading to the extrusion of several small cytoplasmic fragments, was sometimes also observed in early oocytes, we decided to culture both early and late oocytes in CD containing M16 medium up to 5 hours after activation. Such a treatment prevents fragmentation and extrusion of "a second polar body". While swelling, the introduced embryonic stem cell nuclei moved towards the center of the oocytes (in CD-free medium). However, some late oocytes remained located close to the inner surface of an egg membrane.
• a cytological analysis of reconstituted oocytes revealed two different patterns of remodelling of embryonic stem cell nuclei.
• the swelling of embryonic stem cell nuclei was accompanied by decondensation of chromatin and formation of a chromatin net and nucleoli-like structures characteristic of the pronuclear stages.
• the introduced nuclei were darkly stained (regardless of size, although never exceeding the pronuclear size).
• the nucleoli were located near the nuclear envelope, which, in these remodelled nuclei, was not smooth (as in the above mentioned group) but more or less crenated.
• embryonic stem cell nuclei In enucleated early two-cell embryos, embryonic stem cell nuclei either remained unchanged or underwent the first stage of remodeling. In almost half of the enucleated early 2-cell embryos, the embryonic stem cell nuclei swelled and became oval-shaped (rather than round as was observed in nuclear transfer oocytes). They also stained rather darkly. Their size did not exceed the size of half-blastomere nuclei. They were usually about one-third smaller. The cytological analysis of mid-late reconstituted 2-cell embryos was not undertaken. They were immediately transferred to the oviducts and checked either on the next day (immature females - see below) or were left for postimplantation development (pseudopregnant females).
• 3- or 4-cell embryos 3- or 4-cell embryos.
• reconstituted oocytes were cultured in vitro for 24 hours, in which time they underwent a cleavage division.
• binucleate cells In at least four blastocysts from early transfer oocytes and five derived from late oocytes, binucleate cells were found among the inner cell mass cells (binucleate cells indicate tetraploid embryos). In such binucleate cells, two diploid nuclei can form, during the next cleavage division, one tetraploid nucleus. Thus, in the developing embryo, some cells will be diploid and some will be tetraploid. In mammals, diploid/tetraploid mosaics do not develop normally.
• the applied electric field activated only 4.7% (2/42) of early and 10% (5/51) of late oocytes.
• the ethanol treatment appeared to be a highly effective activation agent.
• Five-six hours after ethanol activation and culture 65.6% (40/61) of early and 86.2% (50/58) of late oocytes were activated.
• the applied electric field does not affect the development of early and late 2-cell embryos. Out of 19 late and 21 early 2-cell embryos which were treated with the same electric field parameters as the experimental embryos and transferred to pseudopregnant recipients, 13 and 15 young were born respectively.
• Mouse embryonic stem cells are the only known cell type which retain pluripotency in vitro. They also appear to be fully totipotent, since it is possible to obtain live born mice originating entirely from embryonic stem cells (Nagy, A. et al, Development 770:815-821 (1990)). The question of whether the totipotency of whole embryonic stem cells extends to the cell nucleus was addressed in these studies. In some mammalian species, such as rabbit, sheep, and cattle, the totipotency of isolated blastomeres and their nuclei is equivalent, at least, to the 8-cell stage (for review see: Modlinski, J.A. et al., In Future Aspects in Human In Vitro Fertilization, eds.
• the donor nucleus should initially undergo a sequence of changes, referred to as "remodelling" (Czolowska, R. et al, J. Cell Sci. 69:19-34 (1984)).
• the changes result in morphological and metabolic resemblance to a pronucleus.
• the first indication of remodelling is the swelling of the donor nucleus in amphibians (Gurdon, J.B., Adv. Morphol. 4:1-41 (1964); Gurdon, J.B., J. Embryol. Exp. Morph. 20:401-414 (1968)); and in mammals (mouse: Czolowska, R. et al , J. Cell. Sci.
• a swelling of embryonic stem cell nuclei prior to formation of pronucleus-like structures was observed in about two-thirds of early, and in the majority of late nuclear transfer oocytes. This suggests that until 10 hours after ovulation (22 hours after hCG injection), the oocyte cytoplasm is generally still capable of controlling pronuclear formation and remodelling of foreign nuclei.
• the abnormal swelling of some embryonic stem cell nuclei, prior to the formation of gigantic pronuclei-like structures indicates that a certain equilibrium in protein exchange, probably present during normal pronuclear growth, is disturbed and, thereby, cytoplasmic control over nuclear remodelling is lost.
• chromosomes of free chromatin induce an actin-rich filamentous layer overlaid by a smooth cell membrane in the vicinity of the chromosomes.
• Szollosi et al. (Eur. J. Cell Biol, 42: 140-151 (1986)) and Soltynska et al. (Biology of the Cell 57: 135-142 (1986)) observed similar cortical changes over the thymocyte chromatin in oocyte-thymocyte hybrids. It appears that in embryonic stem cell-enucleated oocyte hybrids, the oocyte cytoplasm can control the donor nuclei and the denuded embryonic stem cell chromatin can induce the cortical layer which cuts off the second polar body.
• haploid embryos can provoke, after implantation, only a decidual reaction (rev'd by Kaufmann, M.H., in Early Mammalian Development: Parthenogenetic Studies, London: Cambridge University Press (1983); Ozil, J.P., Development 709:117-128 (1991). Extrusion of the second body was more evident with embryonic stem cell nuclear transfer to early oocytes.
• Late oocytes soon after fusion and activation, usually started to cleave abnormally, fragmented, or extruded small cytoplasmic fragments.
• the mitotic spindle may become translocated towards the oocyte center. This results in immediate or abnormal cleavages after activation (rev. by Kaufmann, M.H., Early Mammalian Development: Parthenogenetic Studies, London, Cambridge University Press (1983). This was not the case in the studies herein, however. No rapid movement was observed immediately after transfer of embryonic stem cell nuclei into late vs. early oocytes. It is more likely that during aging, changes occur in the oocyte membrane and in the cortical layer which make it more fragile and sensitive to activation factors.
• Embryonic stem cell nuclei introduced into 2-cell embryos were placed in a foreign cytoplasmic environment.
• the appearance of a novel group of 68-73 x 10,000 Mr proteins, whose synthesis is inhibited by alpha amanitin, is thought to mark the starting point of transcriptional activation of the embryonic genome (Flach, G. et al, EMBO
• TRC transcription-requiring complex
• 8-cell stage nuclei were introduced into enucleated 2-cell embryos, they could support preimplantation (Howlett, S.K. et al, Development 707:915-923 (1987)), early postimplantation (Robl, J.M. et al, Biol. Reprod. 34:133-139 (1986)), and could even produce live-born young
• the nuclear remodelling of embryonic stem cell nuclei introduced into enucleated early 2-cell blastomeres was expressed by their swelling and consequent formation of oval-shaped nuclei, approximately one-third smaller than the size of half blastomere nuclei.
• the donor nuclei were not usually able to pass the first steps of morphological remodelling. This suggests that in early 2-cell embryos, a certain pool of proteins (some probably released in to the cytoplasm during the first mitotic division) are still available, whereas in nonenucleated embryos, their acquisition by the donor embryonic stem cell nuclei is suppressed by the resident blastomere nuclei.
• embryonic stem cell nuclei are not handicapped by the competition for these cytoplasmic components. Although the behavior of embryonic stem cell nuclei in late 2-cell embryos was not monitored, it is probable that in such embryos that are already entering the M phase, the donor nuclei undergo chromosome condensation and after completion of mitosis begin reprogramming in full synchrony with the recipient blastomere next cell cycle. In the case that G2 nuclei are introduced, this model might be optimal for further development of reconstituted embryos. Proper synchrony between the nucleus and cytoplasm appears important in the development of reconstituted embryos.
• binucleate cells were found, indicating karyokinesis without cytokinesis.
• a presence of binucleate cells can result in formation of diplo-tetraploid mosaics. Tetraploid embryos (as well as octoploid, Waksmudzka & Modlinski, unpublished) can implant at a high rate but they rarely form embryonic structures (Snow, H.H.L., J. Embryol Exp.
• Embryonic stem cells are, however, different than the other mammalian embryonic cells hitherto used in nuclear transfer experiments. They are derivatives of the inner cell mass of very late blastocysts. In early blastocysts, the inner cell mass still retains a potential to differentiate into trophectodermal cells (Hogan, B. et al, J. Embryol Exp. Morph., 45:93-105 (1978); 62:379- 394 (1981)). During transition from early to late blastocyst stage, the inner cell mass cells with trophectodermal potential are gradually eliminated by apoptosis, i.e.
• blastocelic fluid Pulce, G.B. et al, Proc. Natl Acad. Sci. U.S.A., 86:3654- 3658 (1989); Parchment, R.E. et al, Differentiation, 43:51-58 (1990); rev'd by Parchment, R.E., Int. J. Dev. Biol 37:75-83 (1993)). Therefore, in late blastocysts, the remaining inner cell mass cells lack the ability to form trophectoderm and are able to differentiate only into embryonic tissues
• mice embryonic stem cells can contribute to the fetus and extraembryonic mesoderm, while the ability to colonize trophectoderm is substantially restricted (Beddington, R.S.P. et al, Development, 705:733-738 (1989)).
• embryonic stem cells are deficient in trophectodermal potential and mainly retain the capability of differentiating into embryonic lineages. If this is the case, then after transfer into an egg-cell cytoplasm, full trophectodermal potential should be regained.

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