Classifications

• • 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
  • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
  • C12N15/877—Techniques for producing new mammalian cloned embryos
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • 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
  • C12N2517/00—Cells related to new breeds of animals
  • C12N2517/04—Cells produced using nuclear transfer

Definitions

• a method for the production of an animal (offspring) by the process of nuclear transfer may be produced using donor genetic material in cells taken directly from any stage of an animal, such as an embryo, foetus or adult or from cell cultures established from material from any stage of an animal, such as embryonic, foetal or adult material.
• the process may be used to introduce genetic modifications into the resultant offspring by genetic manipulation and selection of the cells to act as nuclear donors prior to embryo reconstruction.
• the process may be used to produce animals containing multiple genetic modifications by a number of routes including but not limited to; i) introduction of multiple genetic modifications concomitantly into the cultured cell population ii) repeated rounds of transfection and selection of the cultured cell population iii) repeated rounds of transfection, selection, nuclear transfer and re-isolation of a cell population from the embryo, foetus, juvenile or adult animal so formed iv) or any combination thereof.
• the process may be used to de-differentiate cells (including embryonic, foetal or adult somatic cells) for the production of undifferentiated cells, Embryonic Stem cells, other stem cell populations, germ cells or their derivatives or differentiated somatic cell populations preferably from embryonic foetal or adult stages of nuclear transfer reconstructed embryos.
• de-differentiate cells including embryonic, foetal or adult somatic cells
• the technique of nuclear transfer allows the production of offspring by the reconstruction of an animal embryo (which throughout this text includes all concepts of an animal embryo such as an oocyte, egg, zygote or an early embryo).
• Genetic material from a donor cell is transferred to a suitable recipient cell from which the nuclear or genomic genetic material has been removed.
• donor genetic material was taken from undifferentiated cells or blastomeres from early embryos.
• development has been obtained using donor genetic material from differentiated cells maintained in culture and isolated from embryonic (Campbell et al.
• NT nuclear transfer
• the present invention is applicable to all stages of animals (cells, tissues, embryos and whole animals), in particular animals of an ungulate species including, a cow, bull, pig, goat, sheep, camel or water buffalo, as well as a mouse, rat or other rodent, and also lagomorph animals, such as a rabbit.
• animals include, a cow, bull, pig, goat, sheep, camel or water buffalo, as well as a mouse, rat or other rodent, and also lagomorph animals, such as a rabbit.
• the invention does not relate to human reproductive cloning. It does cover human tissue cells and where applicable, human embryos, in particular those embryos under 14 days old.
• oocytes arrested at metaphase of the second meiotic division have been used as cytoplast recipients.
• the donor genetic material has been introduced into the recipient cell cytoplasm by the processes of 1) cell fusion 2) injection of intact cells, lysed cells or nuclei. Transfer of genetic material may occur either at the time of activation, preceding activation (see patent applications WO 97/07669 and WO 97/07668 or following activation (Campbell et al., Biol. Reprod. 49 9330942 (1993); Campbell et al Biol. Reprod. 50 1385-1393 (1994)).
• the Recipient Cell or Cytoplast The Recipient Cell or Cytoplast.
• Oocytes fertilised zygotes and two cell embryos have been used as cytoplast recipients for nuclear transfer.
• oocytes arrested at metaphase of the second meiotic division also termed unfertilised eggs
• the genetic material is arranged upon the meiotic spindle and is easily removed using mechanical means.
• the subject matter of the present invention is to produce a pronucleus-like structure (including a pronucleus) by transfer of a suitable nucleus, preferably to an enucleated Metaphase II oocyte.
• the pronucleus-like structure thus formed will contain factors, which are generally removed during zygote enucleation.
• This pronucleus-like structure is then used as a donor of genetic material for a second nuclear transfer embryo reconstruction. Nuclei or cells of any cell cycle stage may be used as donors of genetic material preferably however; the cell cycle stage of the recipient cytoplast must be controlled accordingly .
• the genetic material When using zygotes, the genetic material may be removed by mechanical aspiration of both pronuclei. Dependent upon species, in order to facilitate visualisation of the pronuclei the zygotes may be centrifuged prior to enucleation.
• the cytoplast In addition to the transfer of donor genetic material from the karyoplast to the cytoplast, the cytoplast must be stimulated to initiate development. When using a fertilised zygote as a cytoplast recipient development has already been initiated by sperm entry at fertilisation. -When using Mil oocytes as cytoplast recipients the oocyte must be activated by other stimuli.
• Nuclear transfer reconstructed embryos may be cultured in vitro to a stage suitable for transfer to a fmal recipient using any suitable culture medium or culture process.
• embryos may be cultured in vivo in the ligated oviduct of a suitable host animal (in general sheep) until a stage suitable for transfer to a final surrogate recipient is reached.
• Embryos from cattle, sheep and other species may be cultured in a trans species recipient, for simplicity a sheep provides a suitable recipient for bovine, ovine and porcine species.
• a sheep provides a suitable recipient for bovine, ovine and porcine species.
• In order to prevent mechanical damage or attack by macrophages to the reconstructed embryos whilst in the oviduct of the temporary recipient it is usual to embed the embryos in a protective layer of agar or similar material.
• the in vitro culture systems presently available for porcine embryos support development to the blastocyst stage, however, the frequency of live births from such embryos is low (Machaty et al., Biology of Reproduction, (1998) 59, 451-455).
• the first nuclear transfer reconstructed embryo may be cultured by any of these methods until the formation of a pronuclear-like body occurs and the embryo is used for the second nuclear transfer reconstruction. In practice it is presently preferred that this first culture period is carried out using a suitable medium in vitro.
• one aspect of the invention provides a method of reconstituting an animal embryo. The process comprising transferring a nucleus into a first oocyte followed by removing and transferring said nucleus from said oocyte to a further recipient cell.
• the nucleus transferred from the first oocyte is in the form of a pronucleus-like structure.
• the second recipient cell is an oocyte or an enucleated fertilised zygote.
• the first step of this procedure is to produce a pronucleus-like structure by transfer of a donor nucleus to an oocyte.
• the oocyte is a mature metaphase II oocyte (unfertilised egg), which has been previously or is simultaneously or subsequently activated, or an activated Mil oocyte (the activation is usually by physical or chemical input).
• An example of the first step is shown in Figure 1 (for simplicities sake, the use of an activated recipient in Figure 1 is not shown, but this is documented in Table 1).
• the pronucleus-like strucure generated by the first step is removed from the first reconstructed embryo and transferred to an oocyte or to an activated parthenote or enucleated fertilised zygote ( Figure 1).
• the transfer is to an oocyte, preferably the pronucleus-like structure is removed from the first reconstructed embryo and transferred to an oocyte which is an enucleated Mil oocyte prior to. concomitant with or following activation.
• the reconstructed embryo may be transferred directly to a final surrogate recipient for development to term.
• the donor genetic material for embryo reconstruction can be provided by any differentiated, partially differentiated or undifferentiated cell taken directly from any stage in an animal development, such as an embryo, foetus, juvenile or adult animal or any cell line derived from such.
• the cell cycle stage of the donor nucleus has been shown to be critical for development.
• a cell at any stage of the cell cycle can be used as donor of genetic material for the first reconstruction process.
• the combination of cell cycle stages of the donor nucleus and the recipient cytoplasm will vary dependent upon the cell cycle stage of the cell to be used as donor of genetic material at the time of transfer.
• the donor genetic material and the recipient cells are not required to be from the same animal species, although this may be preferred.
• Any cell population obtained directly from any stage in the life of an animal may be used as a donor genetic material, alternatively a cell from any population may be used (for example an undifferentiated, partially differentiated or differentiated cell population) derived from any stage (for example embryo, foetus, juvenile or adult animal) and maintained in culture or any cell (for example undifferentiated, partially differentiated or differentiated cell) resulting during culture of primary isolates or as a result of treatment of the cultured cell population with hormones, growth factors or chemicals which alter the differentiated state.
• the cell cycle stage of cells maintained in culture may be manipulated to ensure coordination with the recipient cytoplast using any means available to any one skilled in the art.
• the following examples are non-limiting and are merely representative of various aspects of the art.
• Suitable methods include but are not restricted to;
• V Quiescence induced by addition of a specific growth factor or other substance.
• V Quiescence induced by any physical means such as heat shock, hyperbaric pressure or treatment with chemical compounds which induce a heat shock or stress response e.g puromycin, azacytidine.
• Donor nuclei may be arrested in the Gl phase of the cell cycle by treatment with any chemical, hormone, growth factor or other substance which causes arrest or by any physical stress which induces cell cycle arrest i.e. induction of a heat shock response as a result or elevated temperature or treatment with compounds which induce such a response i.e. puromycin, azacytidine.
• Gl phase cells may be obtained by synchronisation of the cell population by any means and selection of Gl cells at an appropriate time.
• cells may be arrested in the M-phase of the cell cycle by treatment with microtubule disrupting drugs such as Colcemid or Nocodazole, mitotic cells may then be selected from the population by shake-off and placed into a separate culture vessel Gl cells may then be selected at an approp ⁇ ate time following shake off.
• mitotic cells may also be selected from an unsynchromsed population Gl cells may be en ⁇ ched by causing cells to exit the growth cycle and arrest in a quiescent or GO phase (as above) and then restimulatmg growth by re- addition of the restricting factor Gl cells can then be selected at a time following restimulation to be found by expenmentation for each individual cell type/population.
• This technique may be combined with any technique which then arrests cells m either the Gl phase of the cell cycle, or at the Gl /S-phase border i.e treatment with hydroxyurea or aphidicolm
• S-phase cells can be selelOcted or enriched for by treatment with any chemical agent which interferes with any point of the replication procedure e.g aphidicolm which inhibits chain elongation in a concentration dependent manner, Hydroxyurea which inhibits the enzyme ⁇ bonucleotide reductase Cells synchronised in any other stage of the cell cycle may be released and the timing of S-phase after release determined expenmentally.
• any chemical agent which interferes with any point of the replication procedure e.g aphidicolm which inhibits chain elongation in a concentration dependent manner
• Hydroxyurea which inhibits the enzyme ⁇ bonucleotide reductase Cells synchronised in any other stage of the cell cycle may be released and the timing of S-phase after release determined expenmentally.
• these two processes may be combined or any other selection or synchronisation procedure or combination thereof utilised
• G2 phase cells may be selected from a growing population that has been synchronised by any physical, chemical or selective method. Alternatively G2 cells may be en ⁇ ched for by treatment of a synchronised or unsynchromsed cell population with any agents that specifically arrest the cell cycle following the completion of S-phase and p ⁇ or to the onset of mitosis M-phase
• Mitotic cells may be selected from an unsynchromsed population using any means possible including but not being limited to, mitotic shake off, elutriation, FAC's (fluorescence activated cell sorting).
• cells may be arrested in mitosis by treatment with microtubule disrupting agents such as colcemid or nocodazole, by treatment with DMAP or any other serine threonine protein kinase inhibitors or by any other chemical or physical means which disrupts normal growth progression and causes arrest in mitosis.
• Unsynchromsed cell populations may be obtained from any embryonic foetal or adult tissue and cultured in vitro or any cell removed directly from an embryo, foetus, juvenile or adult animal.
• An unsynchronised cell is defined as a cell, the cell cycle stage of which is unknown at the time of transfer to a recipient cytoplast.
• Oocytes to act as suitable cytoplast recipients can be obtained using a variety of techniques and protocols, these include but are not limited to
• oocytes are harvested by flushing from the oviduct in calcium magnesium free phosphate buffered saline (PBS) (or other suitable medium devoid of calcium and magnesium ions) containing 1.0% foetal calf serum (FCS).
• PBS calcium magnesium free phosphate buffered saline
• FCS foetal calf serum
• in vitro matured oocytes are harvested and transferred to suitable medium devoid of calcium and magnesium ions (for example M2 medium prepared without addition of calcium and magnesium ions).
• suitable medium devoid of calcium and magnesium ions
• the chosen medium may be supplemented with FCS, BSA or other protein source.
• Oocytes are denuded of cumulus cells and enucleated as previously described (Campbell et al, 1993.1994) with the exception that calcium free medium, although not essential is preferred for all procedures.
• nucleus may be introduced by manual injection of the donor cell into the enucleated oocyte (Ritchie and Campbell, J. Reproduction and Fertility Abstract Series No. 15, p60) or by using a commercially available piezzo injection apparatus as demonstrated in the mouse (Wakayama et al, 1998). The timing of these events is dependent upon the species.
• the protocols outlined in this application define the use of in vivo and in vitro matured ovine, bovine and porcine oocytes coupled to the- use of in vitro or in vivo produced zygotes. The procedure is not limited to these defined protocols.
• Mamred oocytes may be obtained by any of the procedures outlined above. The genetic material may be removed from the oocyte as previously described. Oocytes may be activated by treating the mamred oocytes with any physical or chemical stimulus or combination thereof, which causes activation and stimulates development. The timing of application of the activation stimulus will vary somewhat upon both the species and the method of mamration. Fertilised zygotes may be obtained by in vitro fertilisation of mamred oocytes obtained from any of the above sources or alternatively recovered ex vivo following mating or Al using stimulated or non-stimulated donor animals.
• Donor cells at different stages of the cell cycle may be used for embryo reconstruction.
• the cytoplast recipient used and any additional treatments required will vary somewhat, dependent upon the cell cycle stage of the donor and recipient cells. Table 1 gives a series of possible combinations.
• any method may be used which results in the production of one or more pronucleus-like structure, preferably of normal diploidy or tetraploidy.
• a pronucleus-like structure (preferably identified as a swollen form of the nucleus) from the first nuclear transfer reconstructed embryo is transferred to an enucleated Mil (prior to, concomitant with or following activation) or to an activated parthenote or fertilised zygote which have been enucleated by removal of the pronuclei.
• an enucleated fertilised zygote is the preferred choice.
• the zygote to act as cytoplast recipient for the second nuclear transfer may be produced in vivo or in vitro, although in practice the use of in vivo produced zygotes is the method of choice at present.
• any cell resulting from development of the first nuclear transfer embryo or cultured cell population derived from this embryo may be used as a donor of genetic material for production of the second nuclear transfer embryo.
• Oocytes obtained from any in vitro or in vivo source and subsequently mamred in vitro, or oocytes mamred in vivo may be used as recipients for the second nuclear transfer following enucleation and subsequent activation or activation and subsequent enucleation.
• the recipient cytoplast for the second nuclear transfer is prepared from a fertilised zygote.
• the zygote required for production of a cytoplast recipient for the second nuclear transfer may be obtained from any suitable source which may include but is not limited to; In vivo produced fertilised zygotes that are recovered from a suitable donor animal. Zygotes may be produced by natural mating, by natural mating following superovulation or by Al using fresh or frozen semen.
• fertilised zygotes may be produced in vitro.
• fertilisation may be carried out on oocytes mamred in vivo from suitable donors with or without superovulation or by using oocytes mamred in vitro following recovery from slaughterhouse material or aspiration of follicles in vivo.
• the zygote for the second nuclear transfer is obtained following maturation and fertilisation in vivo.
• Enucleation of zygotes Following successful fertilisation the male and female chromatin in the newly formed zygote decondenses and forms two pronuclei.
• the genetic material may be removed at any point following fertilisation. However, in practice the present method of choice is to remove the genetic material after formation of the pronuclei. Due to the nature of the cytoplasm it is often difficult to visualise and remove the pronuclei from a fertilised zygote. Visualisation of the pronuclei may be aided by centrifugation of the zygotes prior to their manipulation, although this is not essential.
• Preparation of a karyoplast donor from first nuclear transfer embryo The pronucleus/i formed in the first nuclear transfer reconstructed embryos must be removed and transferred to the second cytoplast recipient.
• the embryos are preferably centrifuged. Aspiration of a pronucleus surrounded by cytoplasm and enveloped in a cell membrane is then carried out, preferably by microsurgery.
• the embryo may be rendered more pliable by treatment with one or more chemical compounds, which disrupt the cytoskeleton e.g. Cytochalasin B, or Nocodazole.
• the centrifuged embryo is preincubated in the agent/s of choice and then transferred to a manipulation chamber. Using a fine glass pipette a karyoplast is aspirated which contains a pronucleus.
• the karyoplast formed from the first nuclear transfer embryo is used to transfer the genetic material now contained within the pronucleus like structure to the enucleated second recipient cytoplast. Any means of transfer including fusion of the karyoplast to the cytoplast by viral, chemical or electrical means may be used or alternatively the pronucleus may be injected into the recipient cell. Due to the size of the pronucleus it is preferred but not essential that the genetic material is transferred by the process of cell fusion. For cell fusion the karyoplast is transferred below the zona pellucida of the second cytoplast recipient and placed into contact with the enucleated zygote. Any physical, chemical or viral means of inducing cell fusion may then be applied.
• the couplet is placed into a suitable medium in a suitable fusion chamber.
• a DC electrical pulse is then passed through the couplet at right angles to the plane of contact between the cytoplast and karyoplast.
• An AC electrical pulse may first be used to align and/or increase contact between the cytoplast and karyoplast.
• the duration strength and timing of the electrical pulse/s all affect the frequency of fusion and can be optimised by experimentation. Culture of second nuclear transfer embryos.
• reconstructed embryos may be cultured in the ligated oviduct of a suitable, synchronised, intermediate recipient for sufficient time to reach a developmental stage suitable for transfer to a final surrogate recipient.
• the second nuclear transfer reconstructed embryo can be transferred directly to the final surrogate recipient and pregnancy maintained by hormonal treatment (see below).
• Reconstructed embryos may be cultured by any means in vitro or in vivo to any stage suitable for transfer to a synchronised final recipient for development to term.
• reconstructed embryos may be transferred as soon as possible to a synchronised recipient for development to term.
• the latter method is presently the method of choice in porcine species.
• induction and maintenance of pregnancy in the final recipient can be achieved by either of the following procedures; At the time of transfer in vivo produced embryos at the same developmental stage as the nuclear transfer reconstructed embryos may be transferred with the nuclear transfer.
• the recipient may be treated with a combination of hormones in order to maintain pregnancy (Christenson et al, J. Animal Science 1971 ; 32:282- 286).
• transgenic refers broadly to any animal stage whose germ line has been the subject of technical intervention by recombinant DNA technology. This includes introduction of genetic material from another species as well as an animal in whose germ line an endogenous gene has been deleted, duplicated, activated or modified.
• the genetic material can be modified at any stage in the double nuclear transfer methodology. Most preferably, the recombinant intervention is carried out before the first nuclear transfer step. The recombinant intervention may be carried out between the first and second nuclear transfer steps.
• the genetic material is preferably modified as part of a cell line modification or taken from a previously modified animal.
• a level of selection can be introduced to identify donor nuclei for the nuclear transfer step.
• recombinant intervention can be carried out after the first nuclear transfer step and before the second.
• the cell obtained may be genetically modified by the same or a different method.
• Suitable recombinant technology for genetically modifying nuclear material is well known in the art. It includes methods as discussed and disclosed in WO97/07669. WO97/07668, US5,612,205, US5.721.367. WO99/01164, WO99/21415, US 5,945,577 and WO98/37183.
• the present invention includes the use of unenucleated oocytes as cytoplast recipients for embryo reconstruction by nuclear transfer.
• the chromatin of cells arrested in GO and poised to undergo differentiation may be more amenable to respond to a variety of signals. Alternatively, it may merely be a 'more null' state that allows the chromatin to respond to oocyte maternal cytoplasmic factors controlling gene expression after nuclear transfer. This hypothesis is supported by previous observations in the mouse using embryonic blastomeres as nuclear donors (see above). In these reports, donors in late G2-phase M-phase and nuclei in early Gl -phase (in which chromatin decondensation is incomplete) have a greater developmental potential than late Gl, S or early G2 donors.
• Nuclei in the G2 or M-phases of the cell cycle can be transferred to a maturing oocyte either I) at a point following metaphase of the first meiotic division i.e. between MI and Mil or ii) at MIL At Mil there would be 2 spindles present 1 from the maternal DNA and the other from the transferred DNA.
• Activation of the recipient oocyte can be prevented by methods known in the art i.e. removal of calcium from the culture, manipulation or fusion medium.
• the transferred chromatin which is condensed into chromosomes may then be allowed to 'be conditioned' in the recipient cytoplasm for a period dependent upon the species and the age of the recipient oocyte.
• the maternal chromatin may be removed following formation of an intact spindle containing the donor chromatin, or alternatively following activation of the reconstructed oocyte. Activation may be achieved by methods common to the art with the exception that spindle integrity must be maintained.
• the transferred DNA undergoes a pseudo- mitotic/meiotic division and extrudes a polar body thus giving a diploid embryo.
• the extrusion of the polar body could be prevented with compounds common to the art e.g. Cytochalasin B and the resulting zygote would have 2 diploid pronuclei each of which could be used for transfer to an enucleated zygote or an activated enucleated oocyte.
• the original oocyte or maternal DNA could be selectively removed following identification by a number of means
• transgenic donor cells which contain a specific marker gene driven by a GO specific promotor sequence.
• Transfer of the donor nucleus can be achieved as previously described using electrical, chemical or virally induced fusion or injection by any means including the use of a piezzo.
• Enucleation following transfer of the donor nucleus can be achieved by any methods known to the art. The timing of the various procedures will vary between species and be dependent upon the source of oocytes and will be determined experimentally.
• the donor cells should be placed in a suitable DNA specific fluorochrome to stain the DNA - i.e. 1 microgram of Hoescht 3332 for 10 minutes in culture medium followed by incubation in medium lacking the stain. Transfer of the nucleus can be carried out at any time after the appearance of telophase of the 1 st meiotic division which may be observed microscopically by the appearance of a small protuberance on the cell wall of the oocyte which is well known to those skilled in the art.
• Manipulation and reconstruction of the recipient oocytes is preferred in a medium that lacks calcium in order to prevent activation.
• the reconstructed oocytes can then be cultured until the maternal DNA is removed. Sufficient time is allowed for formation of the mitotic spindle (this will vary between species and culture conditions and also on the timing of reconstruction).
• sufficient time is allowed for the oocyte maternal chromatin to reach Mil. Enucleation of the maternal chromatin may be achieved at Mil by brief exposure to UV light for visualisation and identification of the stained chromatin.
• the oocytes may be activated and then exposed to UV for identification of the donor chromatin at any time prior to and following pronuclear formation. Following removal of the maternal chromatin and activation the presumptive zygotes are cultored by any means known to the art to a stage suitable for transfer to a synchronised final recipient for development to term.
• the unenucleated oocyte is preferably used in the first nuclear transfer step. It may be used in the second.
• Feed Altrenogest (Regu-Mate, 18 mg/day, 9 AM) for 5 - 9 days, starting on day 12- 16 of the cycle (day 0 is heat). If no heats are available, feed Altrenogest for 14 days. Inject gilts with Estrumate (IM, 1.0 ml, 9 AM) during the last Alternogest feeding. Inject eCG (Equine Chorionic Gonadotropin, 1500 IU IM) 14 firs (11 PM) after the last Altrenogest feeding. Inject hCG (human Chorionic Gonadotropin,
• Enucleated oocytes were maintained in calcium free medium 10% FCS at 39°C.
• the donor genetic material is introduced into the enucleated oocyte by piezzo aided injection.
• the cytoplast recipients may be centrifuged at 13,000xG for 5 minutes.
• the donor cell is placed into medium containing 12% Poly vinyl Pyrollidone (optional), the cell is drawn into the injection pipette and then injected into the oocyte, the cell membrane may be intentionally damaged by selection of an appropriately sized pipette and/or the application of a burst of piezzo vibration and repeated pipetting.
• the size of the injection pipette is dependent upon the size of the cell.
• transfer of the nucleus may be achieved by cell fusion.
• a single cell was placed into contact with the oocyte by using a glass pipette to transfer the cell through the hole previously made in the zona pellucida.
• the cytoplast/ cell couplet was then transferred into fusion medium (0.3 M D-Sorbitol supplemented withO. lmM Mg SO 4 & 0.05 mM CaCl 2 in H 2 0, osmolality 280mOsM) in the fusion chamber.
• the couplet was manually aligned between the electrodes. Fusion was induced by application of two DC pulses of lkV/cm for 60 ⁇ sec at intervals of 5 sec.
• Couplets were then washed in calcium free medium supplemented with 10% FCS at 37°C and incubated in the same medium under oil at 37°C 5% CO2. 30 minutes prior to activation the couplets were transferred to calcium free medium 10% FCS containing 5 ⁇ M Nocodazole.
• Activation was induced as described below, following activation the reconstructed zygotes were incubated in medium NCSU23, 10% FCS at 37°C 5% CO2 for a further 3 hours. They were then washed 3 times for 5 minutes at 37°C in the same medium without nocodazole and cultured for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction.
• the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• Enucleated oocytes were returned to the maturation medium. At 30 or 42 hours post onset of maturation a single cell was placed into contact with the enucleated oocyte. The couplet was transferred to the fusion chamber (see below) in fusion medium 0.3 M D-Sorbitol supplemented withO. lmM Mg SO 4 & 0.05 mM CaCl 2 in H 2 0, osmolality 280mOsM. Fusion and activation were induced by application of two DC pulses of lkV/cm for 60 ⁇ sec at intervals of 5 sec.
• Couplets were then washed in NCSU23 10% FCS and incubated at 37°C 5% CO2 for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction.
• the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• Enucleated oocytes were activated (as described below) and cultured in NCSU23, 10% FCS at 37°C 5% CO2- After the decay of MPF activity, a single cell was then placed into contact with the oocyte and fusion induced as described below. The couplets were then cultured in NCSU23 10% FCS at 37°C 5 %CO2 for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction. Alternatively, the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• a suitable cytoplast may be prepared by activation of an enucleated oocyte as described in the 'UNIVERSAL RECIPIENT'.
• a cytoplast recipient may be prepared from any in vivo or in vitro produced zygote. After pronuclear formation the zygote may be centrifuged, in a suitable buffered medium, at 13.000xg for 5 minutes, this aids visualisation of the pronuclei but is not essential. The centrifuged zygotes are placed into the mampulation chamber in a suitable medium (as above) and a portion of cytoplasm containing the two pronuclei aspirated using a micropipette.
• Feed Altrenogest (Regu-Mate, 18 mg/day, 9 AM) for 5 - 9 days, starting on d 12-16 of the cycle (day 0 is heat). If no heats are available, feed Altrenogest for 14 days.
• Inject eCG (Equine Chorionic Gonadotropin, 1500 IU IM) 14 hrs (11 PM) after the last Altrenogest feeding.
• Inject hCG human Chorionic Gonadotropin, 1000 IU, IM) 79 hrs after the eCG injection (6 AM). Artificially inseminate 24 and 36 hrs after hCG injection. Collect embryos 50 hrs after the hCG injection (noon).
• the karyoplast formed by aspiration of the pronucleus surrounded by a portion of cytoplasm and membrane bound from the first nuclear transfer embryo is placed below the zona pellucida of the second recipient cytoplast in contact with the cytoplast. Fusion of the karyoplast and cytoplast are carried out as below or by any other means. Alternatively the karyoplast, the pronucleus or the pronucleus surrounded by a portion of cytoplasm may be injected into the second nuclear transfer embryo. Culture of reconstructed embryos.
• Reconstructed embryos may be cultured in vitro using any suitable culture medium and conditions e.g. NCSU medium supplemented with 10%FCS 37°C in a humidified atmosphere of 5.0% CO 2 .
• the reconstructed embryos may be transferred directly to a synchronised final recipient for development to term.
• the transfer of embryos to a synchronised recipient has been determined to support good embryo and foetal development.
• porcine zygotes were sham manipulated to mimic the zona pellucida damage occurring during the nuclear transfer procedure. These manipulated embryos were then transferred to a suitable recipient. Subsequently the recipient was sacrificed and development of the transferred embryos assessed (table 2) .
• Manipulated embryos were able to develop to blastocysts and healthy foetuses.
• oocytes were placed between two parallel electrodes in activation medium 0.3 M D-Sorbitol supplemented withO. lmM Mg SO 4 & 0.05 mM CaCl 2 in
• Activation was induced by application of two DC pulses of lkV/cm for 60 ⁇ sec at intervals of 5 sec. This activation procedure promotes development of parthenogenetic embryos to the blastocyst stage (see Table 3).
• any other suitable medium may be used for example 0.3 M mannitol, O. lmM MgSO4, O.OOlmM CaCl2 in distilled water (Willadsen, 1986). Any suitable activation pulse may be used for instance 1 DC pulse of 1.25 kV/cm for 80 ⁇ s.
• Fusion manipulated embryos were treated in a similar manner with the addition that the contact surface between the enucleated oocyte and the cell was arranged parallel to the electrodes. Fusion was induced by application of an AC. current of 3V for 5 seconds followed by DC pulses of 1.0 kV/cm for 60 ⁇ s at 5 second intervals.
• embryos may be cultored by a variety of techniques known to those skilled in the art including; in vitro culture in a suitable medium or in vivo cultore in the ligated oviduct of a suitable recipient host until a stage for transfer to a final surrogate recipient.
• the second stage reconstructed embryos may be transferred directly to the oviduct of a suitable synchronised final recipient host for development to term (see tables 4 and 5).
• Table 5 Pregnancy outcome following transfer of double nuclear transfer embryos directly to a synchronised final recipient.
• the final recipient in vivo produced embryos at the same developmental stage as the reconstructed embryos may be transferred with the nuclear transfer embryos
• the final recipient may be treated hormonally to maintain pregnancy in the following manner, eCG (1000 IU) on day 9 or day 10 of pregnancy, hCG (500 IU) 3 to 3 5 days later
• FSH ovme follicle-stimulating hormone
• Unfertilised metaphase II oocytes were recovered by flushing from the oviduct at 24-29 hours after GnRH injection using Dulbecco's phosphate buffered salme containing 1 0% foetal calf serum (FCS) maintained at 37°C until use
• oocytes were maintained at 37°C, washed m PBS 1 0% FCS and transferred to calcium free M2 medium containing 10% Foetal Calf Serum (FCS), at 37 °C
• FCS Foetal Calf Serum
• oocytes were placed m calcium free M2 containing 10% FCS, 7.5 ug/ml Cytochalasin B (Sigma) (Cytochalasin B is optional) and 5.0 ug/ml Hoechst 33342 (Sigma) at 37°C for 20 minutes.
• Cytochalasin B Sigma
• Hoechst 33342 Sigma
• a range of protocols are available for the first stage nuclear transfer. Their use is dictated by the cell cycle stage of the donor cell nucleus at the time of transfer.
• a single cell was placed into contact with the oocyte by using a glass pipette to transfer the cell through the hole previously made in the zona pellucida.
• the cytoplast/cell couplet was then transferred into the fusion chamber in 200 ⁇ l of 0.3M mannitol in distilled water, and manually aligned between the electrodes.
• An AC pulse of 5V was applied for 3 seconds followed by 3 D.C. pulses of 1.25kV/cm for 80 ⁇ secs.
• the couplets were then washed in calcium free M2, 10% FCS at 37°C and incubated in the same medium under oil at 37°C 5 % CO2.
• the couplets were transferred to calcium free M2 medium 10% FCS containing 5 ⁇ M Nocodazole. Activation was induced at 32-34 hours post hCG injection as described below. Following activation the reconstructed zygotes were incubated in medium TC199 10% FCS at 37°C 5 % CO2 for a further 3 hours. They were then washed 3 times for 5 minutes at 37°C in the same medium without nocodazole and cultored for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction. Alternatively, the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• Couplets were then washed in TCI 99 10% FCS (the addition of 7.5 ⁇ g/ml Cytochalasin B to this medium is optional) and incubated in this medium for 1 hour at 37°C 5% CO2- Couplets were then washed in TC199 10% FCS and cultored in TC199 10% FCS at
• the donor nucleus may be transferred by either manual or piezzo aided injection .or by any other chemical or physical means of producing cell fusion.
• Enucleated oocytes were activated (as described below) 32-34 hours post hCG injection and then cultored in TC199 10% FCS at 37°C 5% CO2 for 4-6 hours. A single cell was then placed into contact with the oocyte and fusion induced as described below. The couplets were then incubated in TC199 10% FCS 7.5 ⁇ g cytochalasin B for 1 hour at 37°C 5%CO2- Couplets were then washed and cultored in TCI 99 10% FCS at 37°C 5 % CO2 for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction. Alternatively, the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• Unfertilised metaphase II oocytes were recovered by flushing from the oviduct at 24-29 hours after GnRH injection using Dulbecco's phosphate buffered saline containing 1.0% foetal calf serum (FCS) maintained at 37°C until use. Recovered oocytes were fertilised in vitro using techniques available to those skilled in the art. Alternatively superstimulated ewes were mated or fertilised by artificial insemination using fresh or frozen serum. Fertilised zygotes were recovered surgically by flushing from the oviduct in any suitable medium.
• FCS foetal calf serum
• a suitable cytoplast may be prepared by activation of an enucleated oocyte as described in the 'UNIVERSAL RECIPIENT' .
• a cytoplast recipient may be prepared from any in vivo or in vitro produced zygote. After pronuclear formation the zygote may be centrifuged, in a suitable buffered medium, at 13.000xg for 5 minutes, this aids visualisation of the pronuclei but is not essential.
• the centrifuged zygotes are placed into the manipulation chamber in a suitable medium (as above) and a portion of cytoplasm containing the two pronuclei aspirated using a micropipette. RECONSTRUCTION OF THE SECOND STAGE NUCLEAR TRANSFER EMBRYO.
• the karyoplast formed by aspiration of the pronucleus surrounded by a portion of cytoplasm and membrane bound from the first nuclear transfer embryo is placed below the zona pellucida of the second recipient cytoplast in contact with the cytoplast. Fusion of the karyoplast and cytoplast are carried out as below or by any other means. Alternatively the karyoplast, the pronucleus or the pronucleus surrounded by a portion of cytoplasm may be injected into the second nuclear transfer embryo.
• oocytes were placed between two parallel electrodes in 200 ⁇ l of 0.3 M mannitol, O. lmM MgSO4, O.OOlmM CaCl2 in distilled water (Willadsen, 1986). Activation was induced by application of 1 DC pulse of 1.25 kV/cm for 80 ⁇ s.
• fusion manipulated embryos were treated in a similar manner with the addition that the contact surface between the enucleated oocyte and the cell was arranged parallel to the electrodes. Fusion was induced by application of an AC. current of 3V for 5 seconds followed by 3 DC pulses of 1.25 kV/cm for 80 ⁇ s
• fused couplets were double embedded in 1 % and 1.2% agar (DIFCO) in PBS (or any other suitable protective covering material) and transferred to the ligated oviduct of unsynchronised ewes.
• recipient ewes were sacrificed and the embryos retrieved by flushing from the oviduct using PBS 10% FCS.
• Embryos were dissected from the agar chips using 2 needles and development assessed by microscopy. All embryos which had developed to the morula/blastocyst stage were transferred as soon as possible to the uterine horn of synchronised final recipient ewes.
• the second stage reconstructed embryos may be transferred directly to the oviduct of a suitable synchronised final recipient host for development to term.
• Bovine oocytes and zygotes for use as recipients for nuclear transfer embryo reconstruction may be obtained from any source or by any of the methods available to anyone skilled in the art.
• the following examples are non-limiting and are merely representative of various aspects of the present invention.
• Ovaries were obtained from a local abattoir and maintained at 28 - 32°C during transport to the laboratory.
• Cumulus oocyte complexes COC's
• COC's Cumulus oocyte complexes
• the universal containers were placed into a warmed chamber (35°C) and the follicular material allowed to settle for 10 - 15 minutes before pouring off three quarters of the supernatant.
• the remaining follicular material was diluted with an equal volume of dissection medium (TCM 199 with Earles salts( Gibco), 75.0 mg/1 kanamycin, 30.0mM Hepes, pH7.4, osmolarity 280 mOsmols/Kg H2O) supplemented with 10% bovine serum , transferred into an 85mm petri dish and searched for COC's under a dissecting microscope.
• TCM 199 with Earles salts( Gibco) 75.0 mg/1 kanamycin, 30.0mM Hepes, pH7.4, osmolarity 280 mOsmols/Kg H2O
• Complexes with at least 2-3 compact layers of cumulus cells were selected washed three times in dissection medium and transferred into maturation medium (TC medium 199 with Earles salts (Gibco), 75mg/l kanamycin, 30.0mM Hepes.
• Matored oocytes were stripped of cumulus cells 18 hours after the onset of maturation. Denuded oocytes were then washed in calcium free M2 medium containing 10% Foetal Calf Serum (FCS) and maintained in this medium at 37 °C. To remove the chromosomes (enucleation) oocytes were placed in calcium free M2 containing 10% FCS, 7.5 ug/ml Cytochalasin B (Sigma) and 5.0 ug/ml Hoechst 33342 (Sigma) at 37 °C for 20 minutes. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using a 20 ⁇ M glass pipette. Enucleation was confirmed by exposing the aspirated portion of cytoplasm to UV light and checking for the presence of a metaphase plate. Enucleated oocytes were then used for each of the three methods of reconstruction as detailed below.
• a range of protocols are available for the first stage nuclear transfer. Their use is dictated by the cell cycle stage of the donor cell nucleus at the time of transfer.
• Enucleated oocytes were maintained in calcium free M2 10% FCS at 39°C as soon as possible after enucleation a single cell was placed into contact with the oocyte by using a glass pipette to transfer the cell through the hole previously made in the zona pellucida.
• the cytoplast/cell couplet was then transferred into the fusion chamber in 200 ⁇ l of 0.3M mannitol in distilled water. The couplet was manually aligned between the electrodes.
• An AC pulse of 3V was applied for 5 seconds followed by 3 D.C. pulses of 1.25kV/cm for 80 ⁇ secs.
• the couplets were then washed in calcium free M2, 10% FCS at 37°C and incubated in the same medium under oil at 37°C 5%
• the couplets were transferred to calcium free M2 medium 10% FCS containing 5 ⁇ M Nocodazole.
• Activation was induced as described below, following activation the reconstructed zygotes were incubated in medium TC199 10% FCS at 37°C 5 % CO2 for a further 3 hours. They were then washed 3 times for 5 minutes at 37°C in the same medium without nocodazole and cultored for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction.
• the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• Enucleated oocytes were retorned to the matoration medium. At 30 or 42 hours post onset of maturation a single cell was placed into contact with the enucleated oocyte.
• the couplet was transferred to the fusion chamber (see below) in 200 ⁇ l of 0.3 M mannitol, O. lmM MgSO4, O.OOlmM CaCl2 in distilled water. Fusion and activation were induced by application of an AC. pulse of 3V for 5 seconds followed by 3 DC. pulses of 1.25kV/Cm for 80 ⁇ secs. Couplets were then washed in TC199 10% FCS and incubated at 37°C 5 % CO2 for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction. Alternatively, the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• Enucleated oocytes were activated (as described below) 30 or 42 hours post onset of matoration and then cultored in TC199 10% FCS at 37°C 5 % CO2 for 8-10 hours (30 hpm group) or 4-6 hours (42 hpm group). A single cell was then placed into contact with the oocyte and fusion induced as described below. The couplets were then cultored in TC199 10% FCS at 37°C 5 %CO2 for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction. Alternatively, the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• a suitable cytoplast may be prepared by activation of an enucleated oocyte as described in the 'UNIVERSAL RECIPIENT'.
• a cytoplast recipient may be prepared from any in vivo or in vitro produced zygote. After pronuclear formation the zygote may be centrifuged, in a suitable buffered medium, at 13.000xg for 5 minutes, this aids visualisation of the pronuclei but is not essential. The centrifuged zygotes are placed into the manipulation chamber in a suitable medium (as above) and a portion of cytoplasm containing the two pronuclei aspirated using a micropipette.
• the karyoplast formed by aspiration of the pronucleus surrounded by a portion of cytoplasm and membrane bound from the first nuclear transfer embryo is placed below the zona pellucida of the second recipient cytoplast in contact with the cytoplast. Fusion of the karyoplast and cytoplast are carried out as below or by any other means. Alternatively the karyoplast, the pronucleus or the pronucleus surrounded by a portion of cytoplasm may be injected into the second nuclear transfer embryo.
• oocytes were placed between two parallel electrodes in 200 ⁇ l of 0.3
• the second stage nuclear transfer fused couplets were double embedded in 1 % and 1.2% agar (DIFCO) in PBS and transferred to the ligated oviduct of synchronised or unsynchronised ewes.
• recipient ewes were sacrificed and the embryos retrieved by flushing from the oviduct using PBS 10% FCS. Embryos were dissected from the agar chips using 2 needles and development assessed by microscopy. Alternatively the second stage reconstructed embryos may be transferred directly to the oviduct of a suitable synchronised final recipient host for development to term.
• Granulosa cells were harvested from ovarian follicles by needle aspiration of follicular fluid. Animals from which ovaries were collected were prepared identically to those used for oocyte collection (see Oocyte recovery below). Follicles were aspirated by penetrating through the cortex of the ovary using an 18 gauge hypodermic needle attached to a 10 ml syringe. Follicular fluid was collected from follicles between 2 and 5 mm in diameter, was placed into a 15ml conical centrifuge tube, and centrifuged at 1000 rpm for 10 min.
• the cell pellet was re- suspended into DMEM (Gibco), containing 10% FCS (Summit Biotech), NEAA, 2 ng/ml bFGF and 6 ⁇ l/ml gentamycin. Cells were expanded for several days and then cryopreserved.
• Donor cells were plated at 1-5 X 10 4 cells per 35 mm dish and were allowed to reach 100% confluency in DMEM medium supplemented with NEAA (0.1 mM), bFGF (2ng/ml), and 10% fetal calf serum. Contact inhibition was chosen for cell synchronization in order to maximize the number of healthy cells with a diploid DNA content. See cell cycle data for details.
• Cells were harvested by trypsinisation and stored in suspension in a calcium-free phosphate buffered version of NCSU-23 medium at 38.5 °C for 20-120 minutes, prior to use as nuclear donors.
• the granulosa cell population contained a large proportion (7.2%) of structures having a DNA content lower than that consistent with a cell in Gl (they were sub-Gl). These structures are characteristic of cells undergoing apoptosis and senescence, and thus indicated that the granulosa cells did not tolerate serum starvation very well. Synchronization of the cells by contact inhibition (100% confluent) was more effective, given that a higher proportion of cells were in G1/G0 (90.3 %), and there were fewer apoptotic sub-Gl cells in the population (only 1.6%).
• hCG human Chorionic Gonadotropin
• Pregnancy was maintained by using a combination of Pregnant Mare Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (hCG).
• PMSG 1000 IU
• Human Chorionic Gonadotropin was injected IM on dlO of the estrous cycle, dl being the day of estrus.
• Human Chorionic Gonadotropin was injected IM 3 to 3.5 days later, on dl3 of the cycle.
• hCG Human Chorionic Gonadotropin
• Zygotes were collected 52-54 hours after the hCG injection using Dulbecco's phosphate buffered saline containing BSA (4 g/1) .
• oocytes were first placed in calcium free phosphate buffered NCSU-23 medium containing 5 ⁇ g/ml Cytochalasin B (Sigma) (Cytochalasin B is optional) and 7.5 ⁇ g/ml Hoechst 33342 (Sigma) at 38°C for 20 minutes. A small amount of cytoplasm from directly beneath the 1st polar body was then aspirated using a 18 ⁇ M glass pipette. Enucleation was confirmed by exposing the aspirated portion of cytoplasm in the pipette to UV light, and checking for the presence of a metaphase plate.
• Groups of 10-20 oocytes were enucleated and placed into 20 ⁇ l drops of calcium free phosphate buffered version of the NCSU-23 medium at 38°C under mineral oil (SIGMA).
• SIGMA calcium free phosphate buffered version of the NCSU-23 medium at 38°C under mineral oil
• a single cell was placed under the zona pellucida in contact with the enucleated cytoplasm.
• the couplet was transferred to a fusion chamber (model # BT-453, BTX Inc. San Diego, CA) containing 700 ⁇ l of 0.3 M mannitol, O. lmM MgS0 4 , O. lmM CaCl 2 in deionized water. Fusion and activation were induced by application of an AC pulse of 5V for 5 seconds followed by 2 DC pulses of 1.5kV/Cm for 60 ⁇ sec using an ECM2001
• Electrocell Manipulator (BTX Inc., San Diego, CA). Couplets were then washed in bicarbonate buffered NCSU-23 medium, and incubated in this medium for 0.5-1 hour at 38.6°C in a humidified atmosphere consisting of 5 % CO 2 in air. Couplets were then checked for fusion at 300X magnification using an inverted microscope. Fused reconstructed embryos were artificially activated using 2 successive DC pulses of 1.2kV/cm for 60 ⁇ sec each, and cultured overnight.
• the double nuclear transfer procedure refers to the reconstruction of enucleated zygotes on day 2, using nuclei produced by day 1 nuclear transfer.
• Zygote Enucleation Zygotes were centrifuged at 13,000 rpm for 15 min in Biofuge 13 centrifuge. The zygotes were then placed in phosphate buffered NCSU-23 medium containing 5.0 ⁇ g/ml Cytochalasin B (Sigma) at 38°C, and incubated for 20 minutes. incubated for 20 minutes. Fertilized zygotes with 2 pronuclei were used for enucleation.
• an enucleation pipette (25-35 ⁇ m) was inserted through the zona pelucida and into the cytoplasm, so that the opening of the enucleation pipette was close to the pronuclei. Both pronuclei and the polar bodies were aspirated by slowly applying negative pressure on the enucleation line.
• Couplets were then washed with NCSU-23 medium, incubated in this medium for 0.5-1 hour at 38.6°C, 5% CO 2 , and then checked for fusion. Fused couplets were transferred as soon as possible to the oviduct of an estrus-synchronized recipient gilt. Results of nuclear transfer using primary sranulosa cells are summarized in Table 7. Table 7. Nuclear transfer using porcine granulosa cells
• Ultrasound images confirmed the pregnancies at day 29 and day 42 of gestation.
• Bovine oocytes and zygotes for use as recipients for nuclear transfer embryo reconstruction may be obtained from any source or by any of the methods available to anyone skilled in the art.
• the following examples are non-limiting and are merely representative of various aspects of the present invention.
• Ovaries were obtained from a local abattoir and maintained at 28 - 32°C during transport to the laboratory.
• Cumulus oocyte complexes (COC's) were aspirated from follicles 3 - 10mm in diameter using a hypodermic needle (1.2mm internal diameter) and placed into sterile plastic universal containers.
• the universal containers were placed into a warmed chamber (35 °C) and the follicular material allowed to settle for 10 - 15 minutes before pouring off three quarters of the supernatant.
• the remaining follicular material was diluted with an equal volume of dissection medium ( TCM 199 with Earles salts( Gibco), 75.0 mg/1 kanamycin. 30.0mM Hepes. pH7.4.
• osmolarity 280 mOsmols/Kg H2O supplemented with 10% bovine serum . transferred into an 85mm petri dish and searched for COC's under a dissecting microscope. Complexes with at least 2-3 compact layers of cumulus cells were selected washed three times in dissection medium and transferred into matoration medium (TC medium 199 with Earles salts (Gibco), 75mg/l kanamycin, 30.0mM Hepes, 7.69mM NaHCO3, pH7.8, osmolarity
• the oocytes were placed into medoium containing Hoescht 3332 (Sigma) l ⁇ g /ml for 10 minutes ⁇ washed 3 times in maturation medium without Hoescht and retorned to matoration.
• the donor cells were exposed to growth medium containing Hoescht 3332) l ⁇ g /ml for 10 minutes then washed 3 times in growth medium and stored in cultore medium until use.
• Matored oocytes were stripped of cumulus cells 18 hours after the onset of matoration. Denuded oocytes were then washed in calcium free M2 medium containing 10% Foetal Calf Serum (FCS) and maintained in this medium at 37°C.
• FCS Foetal Calf Serum
• oocytes were maintained in calcium free M2 10% FCS at 39°C a single cell was placed into contact with the oocyte by using a glass pipette to transfer the cell through the hole previously made in the zona pellucida.
• the cytoplast/cell couplet was then transferred into the fusion chamber in 200 ⁇ l of 0.3M mannitol in distilled water. The couplet was manually aligned between the electrodes.
• An AC pulse of 3V was applied for 5 seconds followed by 3 D.C. pulses of 1.25kV/cm for 80 ⁇ secs.
• the couplets were then washed in calcium free M2, 10% FCS at 37°C and incubated in the same medium under oil at 37°C 5% CO2 for 60 minutes. Fusion was assessed microscopically. Fused couplets were placed into matoration medium for 2 -20 hours. Alternatively, the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• oocytes were incubated as described above. Following incubation the maternal chromatin was removed. To remove the chromosomes (enucleation) oocytes were placed in calcium free M2 containing 10% FCSat 37 °C. If the 1 st polar body was visible a small amount of cytoplasm from directly beneath the 1st polar body was then aspirated using a 20 ⁇ M glass pipette. If the polar body was not visible the oocyte was exposed briefly 1-2 seconds to UV light to identify the stained donor (or oocyte) chromatin. 2. At 24-45 hours post onset of matoration oocytes were activated as described below.
• Enucleated oocytes were retorned to the matoration medium. At 30 or 42 hours post onset of matoration a single cell was placed into contact with the enucleated oocyte. The couplet was transferred to the fusion chamber (see below) in 200 ⁇ l of 0.3 M mannitol, O. lmM MgSO4, O.OOlmM CaCl2 in distilled water. Fusion and activation were induced by application of an AC. pulse of 3V for 5 seconds followed by 3 DC. pulses of 1.25kV/Cm for 80 ⁇ secs.
• Couplets were then washed in TC199 10% FCS and incubated at 37°C 5% CO2 for a further period of time sufficient for the formation of a pronucleus prior to being used as nuclear donors for the second stage nuclear transfer embryo reconstruction.
• the donor nucleus may be transferred by either manual or piezzo aided injection or by any other chemical or physical means of producing cell fusion.
• oocytes were placed between two parallel electrodes in 200 ⁇ l of 0.3 M mannitol, O.lmM MgSO4, O.OOlmM CaCl2 in distilled water (Willadsen, 1986). Activation was induced by application of 1 DC pulse of 1.25 kV/cm for
• nuclear transfer reconstructed embryos may be cultored by any method known to the art these include but are not limited to ;
• Embryos were double embedded in 1 % and 1.2% agar (DIFCO) in PBS and transferred to the ligated oviduct of synchronised or unsynchronised ewes.
• DICO agar
• the reconstructed embryos may be transferred directly to the oviduct of a suitable synchronised final recipient host for development to term.
• embryos may be cultured in vitro by any suitable means to a stage suitable for transfer to a synchronized recipient.

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