Unactivated oocytes as cytoplast recipients of quiescent and non-quiescent cell nuclei, while maintaining correct ploidy

A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

This invention relates to the generation of animals including but not being
 limited to genetically selected and/or modified animals, and to cells
 useful in their generation.
 The reconstruction of mammalian embryos by the transfer of a donor nucleus
 to an enucleated oocyte or one cell zygote allows the production of
 genetically identical individuals. This has clear advantages for both
 research (i.e. as biological controls) and also in commercial applications
 (i.e. multiplication of genetically valuable livestock, uniformity of meat
 products, animal management).
 Embryo reconstruction by nuclear transfer was first proposed (Spemann,
 Embryonic Development and Induction 210-211 Hofner Publishing Co., New
 York (1938)) in order to answer the question of nuclear equivalence or `do
 nuclei change during development?`. By transferring nuclei from
 increasingly advanced embryonic stages these experiments were designed to
 determine at which point nuclei became restricted in their developmental
 potential. Due to technical limitations and the unfortunate death of
 Spemann these studies were not completed until 1952, when it was
 demonstrated in the frog that certain nuclei could direct development to a
 sexually mature adult (Briggs and King, Proc. Natl. Acad., Sci. USA 38
 455-461 (1952)). Their findings led to the current concept that equivalent
 totipotent nuclei from a single individual could, when transferred to an
 enucleated egg, give rise to "genetically identical" individuals. In the
 true sense of the meaning these individuals would not be clones as unknown
 cytoplasmic contributions in each may vary and also the absence of any
 chromosomal rearrangements would have to be demonstrated.
 Since the demonstration of embryo cloning in amphibians, similar techniques
 have been applied to mammalian species. These techniques fall into two
 categories:
 1) transfer of a donor nucleus to a matured metaphase II oocyte which has
 had its chromosomal DNA removed and
 2) transfer of a donor nucleus to a fertilised one cell zygote which has
 had both pronuclei removed. In ungulates the former procedure has become
 the method of choice as no development has been reported using the latter
 other than when pronuclei are exchanged.
 Transfer of the donor nucleus into the oocyte cytoplasm is generally
 achieved by inducing cell fusion. In ungulates fusion is induced by
 application of a DC electrical pulse across the contact/fusion plane of
 the couplet. The same pulse which induces cell fusion also activates the
 recipient oocyte. Following embryo reconstruction further development is
 dependent on a large number of factors including the ability of the
 nucleus to direct development i.e. totipotency, developmental competence
 of the recipient cytoplast (i.e. oocyte maturation), oocyte activation,
 embryo culture (reviewed Campbell and Wilmut in Vth World Congress on
 Genetics as Applied to Livestock 20 180-187 (1994)).
 In addition to the above we have shown that maintenance of correct ploidy
 during the first cell cycle of thee reconstructed embryo is of major
 importance (Campbell et al., Biol. Reprod. 49 933-942 (1993); Campbell et
 al., Biol. Reprod. 50 1385-1393 (1994)). During a single cell cycle all
 genomic DNA must be replicated once and only once prior to mitosis. If any
 of the DNA either fails to replicate or is replicated more than once then
 the ploidy of that nucleus at the time of mitosis will be incorrect. The
 mechanisms by which replication is restricted to a single round during
 each cell cycle are unclear, however, several lines of evidence have
 implicated that maintenance of an intact nuclear membrane is crucial to
 this control. The morphological events which occur in the donor nucleus
 after transfer into an enucleated metaphase II oocyte have been studied in
 a number of species including mouse (Czolowiska et al., J. Cell Sci. 69
 19-34 (1984)), rabbit (Collas and Robl, Biol. Reprod. 45 455-465 (1991)),
 pig (Prather et al., J. Exp. Zool. 225 355-358 (1990)), cow (Kanka et al.,
 Mol. Reprod. Dev. 29 110-116 (1991)). Immediately upon fusion the donor
 nuclear envelope breaks down (NEBD), and the chromosomes prematurely
 condense (PCC). These effects are catalysed by a cytoplasmic activity
 termed maturation/mitosis/meiosis promoting factor (MPF). This activity is
 found in all mitotic and meiotic cells reaching a maximal activity at
 metaphase. Matured mammalian oocytes are arrested at metaphase of the 2nd
 meiotic division (metaphase II) and have high MPF activity. Upon
 fertilisation or activation MPF activity declines, the second meiotic
 division is completed and the second polar body extruded, the chromatin
 then decondenses and pronuclear formation occurs. In nuclear transfer
 embryos reconstructed when MPF levels are high NEBD and PCC occur; these
 events are followed, when MPF activity declines, by chromatin
 decondensation and nuclear reformation and subsequent DNA replication. III
 reconstructed embryos correct ploidy can be maintained in one of two ways;
 firstly by transferring nuclei at a defined cell cycle stage, e.g. diploid
 nuclei of cells in G1, into metaphase II oocytes at the time of
 activation; or secondly by activating the recipient oocyte and
 transferring the donor nucleus after the disappearance of MPF activity. In
 sheep this latter approach has yielded an increase in the frequency of
 development to the blastocyst stage from 21% to 55% of reconstructed
 embryos when using blastomeres from 16 cell embryos as nuclear donors
 (Campbell et al., Biol. Reprod. 50 1385-1393 (1994)).
 These improvements in the frequency of development of reconstructed embryos
 have as yet not addressed the question of nuclear reprogramming. During
 development certain genes become "imprinted" i.e. are altered such that
 they are no longer transcribed. Studies on imprinting have shown that this
 "imprinting" is removed during germ cell formation (i.e. reprogramming).
 One possibility is that this reprogramming is affected by exposure of the
 chromatin to cytoplasmic factors which are present in cells undergoing
 meiosis. This raises the question of how we may mimic this situation
 during the reconstruction of embryos by nuclear transfer in order to
 reprogram the developmental clock of the donor nucleus.
 It has now been found that nuclear transfer into an oocyte arrested in
 metaphase II can give rise to a viable embryo if normal ploidy (i.e.
 diploidy) is maintained and if the embryo is not activated at the time of
 nuclear transfer. The delay in activation allows the nucleus to remain
 exposed to the recipient cytoplasm.
 According to a first aspect of the present invention there is provided a
 method of reconstituting an animal embryo, the method comprising
 transferring a diploid nucleus into an oocyte which is arrested in the
 metaphase of the second meiotic division without concomitantly activating
 the oocyte, keeping the nucleus exposed to the cytoplasm of the recipient
 for a period of time sufficient for the reconstituted embryo to become
 capable of giving rise to a live birth and subsequently activating the
 reconstituted embryo while maintaining correct ploidy. At this stage, the
 reconstituted embryo is a single cell.
 In principle, the invention is applicable to all animals, including birds
 such as domestic fowl, amphibian species and fish species. In practice,
 however, it will be to non-human animals, especially non-human mammals,
 particularly placental mammals, that the greatest commercially useful
 applicability is presently envisaged. It is with ungulates, particularly
 economically important ungulates such as cattle, sheep, goats, water
 buffalo, camels and pigs that the invention is likely to be most useful,
 both as a means for cloning animals and as a means for generating
 transgenic animals. It should also be noted that the invention is also
 likely to be applicable to other economically important animal species
 such as, for example, horses, llamas or rodents, e.g. rats or mice, or
 rabbits.
 The invention is equally applicable in the production of transgenic, as
 well as non-transgenic animals. Transgenic animals may be produced from
 genetically altered donor cells. The overall procedure has a number of
 advantages over conventional procedures for the production of transgenic
 (i.e. genetically modified) animals which may be summarised as follows:
 (1) fewer recipients will be required;
 (2) multiple syngeneic founders may be generated using clonal donor cells;
 (3) subtle genetic alteration by gene targeting is permitted;
 (4) all animals produced from embryos prepared by the invention should
 transmit the relevant genetic modification through the germ line as each
 animal is derived from a single nucleus; in contrast, production of
 transgenic animals by pronuclear injection or chimerism after inclusion of
 modified stem cell populations by blastocyst injection produces a
 proportion of mosaic animals in which all cells do not contain the
 modification and may not transmit the modification through the germ line;
 and
 (5) cells can be selected for the site of genetic modification (e.g.
 integration) prior to the generation of the whole animal.
 It should be noted that the term "transgenic", in relation to animals,
 should not be taken to be limited to referring to animals containing in
 their germ line one or more genes from another species, although many
 transgenic animals will contain such a gene or genes. Rather, the term
 refers more broadly to any animal whose germ line has been the subject of
 technical intervention by recombinant DNA technology. So, for example, an
 animal in whose germ line an endogenous gene has been deleted, duplicated,
 activated or modified is a transgenic animal for the purposes of this
 invention as much as an animal to whose germ line an exogenous DNA
 sequence has been added.
 In embodiments of the invention in which the animal is transgenic, the
 donor nucleus is genetically modified. The donor nucleus may contain one
 or more transgenes and the genetic modification may take place prior to
 nuclear transfer and embryo reconstitution. Although micro-injection,
 analogous to injection into the male or female pronucleus of a zygote, may
 be used as a method of genetic modification, the invention is not limited
 to that methodology: mass transformation or transfection techniques can
 also be used e.g. electroporation, viral transfection or lipofection.
 In the method of the invention described above, a diploid nucleus is
 transferred from a donor into the enucleated recipient oocyte. Donors
 which are diploid at the time of transfer are necessary in order to
 maintain the correct ploidy of the reconstituted embryo; therefore donors
 may be either in the G1 phase or preferably, as is the subject of our
 co-pending PCT patent application No. PCT/GB96/02099 filed today (claiming
 priority from GB 9517780.4), in the G0 phase of the cell cycle.
 The mitotic cell cycle has four distinct phases, G, S, G2 and M. The
 beginning event in the cell cycle, called start, takes place in the G1
 phase and has a unique function. The decision or commitment to undergo
 another cell cycle is made at start. Once a cell has passed through start,
 it passes through the remainder of the G1. phase, which is the pre-DNA
 synthesis phase. The second stage, the S phase, is when DNA synthesis
 takes place. This is followed by the G2 phase, which is the period between
 DNA synthesis and mitosis. Mitosis itself occurs at the M phase. Quiescent
 cells (which include cells in which quiescence has been induced as well as
 those cells which are naturally quiescent, such as certain fully
 differentiated cells) are generally regarded as not being in any of these
 four phases of the cycle; they are usually described as being in a G0
 state, so as to indicate that they would not normally progress through the
 cycle. The nuclei of quiescent G0 cells, like the nuclei of G1 cells, have
 a diploid DNA content; both of such diploid nuclei can be used in the
 present invention.
 Subject to the above, it is believed that there is no significant
 limitation on the cells that can be used in nuclear donors: fully or
 partially differentiated cells or undifferentiated cells can be used as
 can cells which are cultured in vitro or abstracted ex vivo. The only
 limitation is that the donor cells have normal DNA content and be
 karyotypically normal. A preferred source of cells is disclosed in our
 co-pending PCT patent application No. PCT/GB95/02095, published as WO
 96/07732. It is believed that all such normal cells contain all of the
 genetic information required for the production of an adult animal. The
 present invention allows this information to be provided to the developing
 embryo by altering chromatin structure such that the genetic material can
 re-direct development.
 Recipient cells useful in the invention are enucleated oocytes which are
 arrested in the metaphase of the second meiotic division. In most
 vertebrates, oocyte maturation proceeds in vivo to this fairly late stage
 of the egg maturation process and then arrests. At ovulation, the arrested
 oocyte is released from the ovary (and, if fertilisation occurs, the
 oocyte is naturally stimulated to complete meiosis). In the practice of
 the invention, oocytes can be matured either in vitro or in vivo and are
 collected on appearance of the 1st polar body or as soon as possible after
 ovulation, respectively.
 It is preferred that the recipient be enucleate. While it has been
 generally assumed that enucleation of recipient oocytes in nuclear
 transfer procedures is essential, there is no published experimental
 confirmation of this judgement. The original procedure described for
 ungulates involved splitting the cell into two halves, one of which was
 likely to be enucleated (Willadsen Nature 320 (6) 63-65 (1986)). This
 procedure has the disadvantage that the other unknown half will still have
 the metaphase apparatus and that the reduction in volume of the cytoplasm
 is believed to accelerate the pattern of differentiation of the new embryo
 (Eviskov et al., Development 109 322-328 (1990)).
 More recently, different procedures have been used in attempts to remove
 the chromosomes with a minimum of cytoplasm. Aspiration of the first polar
 body and neighbouring cytoplasm was found to remove the metaphase II
 apparatus in 67% of sheep oocytes (Smith & Wilmut Biol. Reprod. 40
 1027-1035 (1989)). Only with the use of DNA-specific fluorochrome (Hoechst
 33342) was a method provided by which enucleation would be guaranteed with
 the minimum reduction in cytoplasmic volume (Tsunoda et al., J. Reprod.
 Fertil. 82 173 (1988)). In livestock species, this is probably the method
 of routine use at present (Prather & First J. Reprod. Fertil. Suppl. 41
 125 (1990), Westhusin et al., Biol. Reprod. (Suppl.) 42 176 (1990)).
 There have been very few reports of non-invasive approaches to enucleation
 in mammals, whereas in amphibians, irradiation with ultraviolet light is
 used as a routine procedure (Gurdon Q. J. Microsc. Soc. 101 299-311
 (1960)). There are no detailed reports of the use of this approach in
 mammals, although during the use of DNA-specific fluorochrome it was noted
 that exposure of mouse oocytes to ultraviolet light for more than 30
 seconds reduced the developmental potential of the cell (Tsunoda et al.,
 J. Reprod. Fertil. 82 173 11988)).
 As described above enucleation may be achieved physically, by actual
 removal of the nucleus, pro-nuclei or metaphase plate (depending on the
 recipient cell), or functionally, such as by the application of
 ultraviolet radiation or another enucleating influence.
 After enucleation, the donor nucleus is introduced either by fusion to
 donor cells under conditions which do not induce oocyte activation or by
 injection under non-activating conditions. In order to maintain the
 correct ploidy of the reconstructed embryo the donor nucleus must be
 diploid (i.e. in the G0 or G1 phase of the cell cycle) at the time of
 fusion.
 Once suitable donor and recipient cells have been prepared, it is necessary
 for the nucleus of the former to be transferred to the latter. Most
 conveniently, nuclear transfer is effected by fusion. Activation should
 not take place at the time of fusion.
 Three established methods which have been used to induce fusion are:
 (1) exposure of cells to fusion-promoting chemicals, such as polyethylene
 glycol;
 (2) the use of inactivated virus, such as Sendai virus; and
 (3) the use of electrical stimulation.
 Exposure of cells to fusion-promoting chemicals such as polyethylene glycol
 or other glycols is a routine procedure for the fusion of somatic cells,
 but it has not been widely used with embryos. As polyethylene glycol is
 toxic it is necessary to expose the cells for a minimum period and the
 need to be able to remove the chemical quickly may necessitate the removal
 of the zona pellucida (Kanka et al., Mol. Reprod. Dev. 29 110-116 (1991)).
 In experiments with mouse embryos, inactivated Sendai virus provides an
 efficient means for the fusion of cells from cleavage-st age embryos
 (Graham Wistar enst. Symp. Monogr. 9 19 (1969)), with the additional
 experimental advantage that activation is not induced. In ungulates,
 fusion is commonly achieved by the same electrical stimulation that is
 used to induce parthogenetic activation (Willadsen Nature 320 (6) 63-65
 (1986), Prather et al., Biol. Reprod. 37 859-866 (1987)). In these
 species, Sendai virus induces fusion in a proportion of cases, but is not
 sufficiently reliable for routine application (Willadsen Nature 320 (6)
 63-65 (1986)).
 While cell-cell fusion is a preferred method of effecting nuclear transfer,
 it is not the only method that can be used. Other suitable techniques
 include microinjection (Ritchie and Campbell, J. Reproduction and
 Fertility Abstract Series No. 15, p60).
 In a preferred embodiment of the invention, fusion of the oocyte karyoplast
 couplet is accomplished in the absence of activation by electropulsing in
 0.3M mannitol solution or 0.27M sucrose solution; alternatively the
 nucleus may be introduced by injection in a calcium free medium. The age
 of the oocytes at the time of fusion/injection and the absence of calcium
 ions from the fusion/injection medium prevent activation of the recipient
 oocyte.
 In practice, it is best to enucleate and conduct the transfer s soon as
 possible after the oocyte reaches metaphase II. The time that this will be
 post onset of maturation (in vitro) or hormone treatment (in vivo) will
 depend on the species. For cattle or sheep, nuclear transfer should
 preferably take place within 24 hours; for pigs, within 48 hours; mice,
 within 12 hours; and rabbits within 20-24 hours. although transfer can
 take place later, it becomes progressively more difficult to achieve as
 the oocyte ages. High MPF activity is desirable.
 Subsequently, the fused reconstructed embryo, which is generally returned
 to the maturation medium, is maintained without being activated so that
 the donor nucleus is exposed to the recipient cytoplasm for a period of
 time sufficient to allow the reconstructed embryo to become capable,
 eventually, of giving rise to a live birth (preferably of a fertile
 offspring).
 The optimum period of time before activation varies from species to species
 and can readily be determined by experimentation. For cattle, a period of
 from 6 to 20 hours is appropriate. The time period should probably not be
 less than that which will allow chromosome formation, and it should not be
 so long either that the couplet activates spontaneously or, in extreme
 cases that it dies.
 When it is time for activation, any conventional or other suitable
 activation protocol can be used. Recent experiments have shown that the
 requirements for parthogenetic activation are more complicated than had
 been imagined. It had been assumed that activation is an all-or-none
 phenomenon and that the large number of treatments able to induce
 formation of a pronucleus were all causing "activation". However, exposure
 of rabbit oocytes to repeated electrical pulses revealed that only
 selection of an appropriate series of pulses and control of the Ca.sup.2+
 was able to promote development of diploidized oocytes to mid-gestation
 (Ozil Development 109 117-127 (1990)). During fertilization there are
 repeated, transient increases in intracellular calcium concentration
 (Cutbertson & Cobbold Nature 316 541-542 (1985)) and electrical pulses are
 believed to cause analogous increases in calcium concentration. There is
 evidence that the pattern of calcium transients varies with species and it
 can be anticipated that the optimal pattern of electrical pulses will vary
 in a similar manner. The interval between pulses in the rabbit is
 approximately 4 minutes (Ozil Development 109 117-127 (1990)), and in the
 mouse 10 to 20 minutes (Cutbertson & Cobbold Nature 316 541-542 (1985)),
 while there are preliminary observations in the cow that the interval is
 approximately 20 to 30 minutes (Robl et al., in Symposium on Cloning
 Mammals by Nuclear Transplantation (Seidel ed.), Colorado State
 University, 24-27 (1992)). In most published experiments activation was
 induced with a single electrical pulse, but new observations suggest that
 the proportion of reconstituted embryos that develop is increased by
 exposure to several pulses (Collas & Robl Biol. Reprod. 43 877-884
 (1990)). In any individual case, routine adjustments may be made to
 optimise the number of pulses, the field strength and duration of the
 pulses and the calcium concentration of the medium.
 In the practice of the invention, correct ploidy must be maintained during
 activation. It is desirable to inhibit or stabilise microtubule
 polymerisation in order to prevent the production of multiple pronuclei,
 thereby to maintain correct ploidy. This can be achieved by the
 application of a microtubule inhibitor such as nocodazole at an effective
 concentration (such as about 5 .mu.g/ml). Colchecine and colcemid are
 other microtubule inhibitors. Alternatively, a microtubule stabiliser,
 such as, for example, taxol could be used.
 The molecular component of microtubules (tubulin) is in a state of dynamic
 equilibrium between the polymerised and non-polymerised states.
 Microtubule inhibitors such as nocodazole prevent the addition of tubulin
 molecules to microtubules, thereby disturbing the equilibrium and leading
 to microtubule depolymerisation and destruction of the spindle. It is
 preferred to add the microtubule inhibitor a sufficient time before
 activation to ensure complete, or almost complete, depolymerisation of the
 microtubules. Twenty to thirty minutes is likely to be sufficient in most
 cases. A microtubule stabiliser such as taxol prevents the breakdown of
 the spindle and may also therefore prevent the production of multiple
 pronuclei. Use of a microtubule stabiliser is preferably under similar
 conditions to those used for microtubule inhibitors.
 The microtubule inhibitor or stabiliser should remain present after
 activation until pronuclei formation. It should be removed thereafter, and
 in any event before the first division takes place.
 In a preferred embodiment of the invention at 30-42 hours post onset of
 maturation (bovine and ovine, i.e. 6-18 hours post nuclear transfer) the
 reconstructed oocytes are placed into medium containing nocodazole (5
 .mu.g/ml) and activated using conventional protocols. Incubation in
 nocodazole may be continued for 4-6 hours following the activation
 stimulus (dependent upon species and oocyte age).
 According to a second aspect of the invention, there is provided a viable
 reconstituted animal embryo prepared by a method as described previously.
 According to a third aspect of the invention, there is provided a method of
 preparing an animal, the method comprising:
 (a) reconstituting an animal embryo as described above; and
 (b) causing an animal to develop to term from the embryo; and
 (c) optionally, breeding from the animal so formed.
 Step (a) has been described in depth above.
 The second step, step (b) in the method of this aspect of the invention is
 to cause an animal to develop to term from the embryo. This may be done
 directly or indirectly.
 In direct development, the reconstituted embryo from step (a) is simply
 allowed to develop without further intervention beyond any that may be
 necessary to allow the development to take place. In indirect development,
 however, the embryo may be further manipulated before full development
 takes place. For example, the embryo may be split and the cells clonally
 expanded, for the purpose of improving yield.
 Alternatively or additionally, it may be possible for increased yields of
 viable embryos to be achieved by means of the present invention by clonal
 expansion of donors and/or if use is made of the process of serial
 (nuclear) transfer. A limitation in the presently achieved rate of
 blastocyst formation may be due to the fact that a majority of the embryos
 do not "reprogram" (although an acceptable number do) If this is the case,
 then the rate may be enhanced as follows. Each embryo that does develop
 itself can be used as a nuclear donor at the 32-64 cell stage;
 alternatively, inner cell mass cells can be used at the blastocyst stage.
 If these embryos do indeed reflect those which have reprogrammed gene
 expression and those nuclei are in fact reprogrammed (as seems likely),
 then each developing embryo may be multiplied in this way by the
 efficiency of the nuclear transfer process. The degree of enhancement
 likely to be achieved depends upon the cell type. In sheep, it is readily
 possible to obtain 55% blastocyst stage embryos by transfer of a single
 blastomere from a 16 cell embryo to a preactivated "Universal Recipient"
 oocyte. So it is reasonable to hypothesise that each embryo developed from
 a single cell could give rise to eight at the 16 cell stage. Although
 these figures are just a rough guide, it is clear that at later
 developmental stages the extent of benefit would depend on the efficiency
 of the process at: that stage.
 Aside from the issue of yield-improving expediencies, the reconstituted
 embryo may be cultured, in vivo or in vitro to blastocyst.
 Experience suggests that embryos derived by nuclear transfer are different
 from normal embryos and sometimes benefit from or even require culture
 conditions in vivo other than those in which embryos are usually cultured
 (at least in vivo). The reason for this is not known. In routine
 multiplication of bovine embryos, reconstituted embryos (many of them at
 once) have been cultured in sheep oviducts for 5 to 6 days (as described
 by Willadsen, In Mammalian Egg Transfer (Adams, E. E., ed.) 185 CRC Press,
 Boca Raton, Fla. (1982)). In the practice of the present invention,
 though, in order to protect the embryo it should preferably be embedded in
 a protective medium such as agar before transfer and then dissected from
 the agar after recovery from the temporary recipient. The function of the
 protective agar or other medium is twofold: first, it acts as a structural
 aid for the embryo by holding the zona pellucida together; and secondly it
 acts as barrier to cells of the recipient animal's immune system. Although
 this approach increases the proportion of embryos that form blastocysts,
 there is the disadvantage that a number of embryos may be lost.
 If in vitro conditions are used, those routinely employed in the art are
 quite acceptable.
 At the blastocyst stage, the embryo may be screened for suitability for
 development to term. Typically, this, will be done where the embryo is
 transgenic and screening and selection for stable integrants has been
 carried out. Screening for non-transgenic genetic markers may also be
 carried out at this stage. However, because the method of the invention
 allows for screening of donors at an earlier stage, that will generally be
 preferred.
 After screening, if screening has taken place, the blastocyst embryo is
 allowed to develop to term. This will generally be in vivo. If development
 up to blastocyst has taken place in vitro, then transfer into the final
 recipient animal takes place at this stage. If blastocyst development has
 taken place in vivo, although in principle the blastocyst can be allowed
 to develop to term in the pre-blastocyst host, in practice the blastocyst
 will usually be removed from the (temporary) pre-blastocyst recipient and,
 after dissection from the protective medium, will be transferred to the
 (permanent) post-blastocyst recipient.
 In optional step (c) of this aspect of the invention, animals may be bred
 from the animal prepared by the preceding steps. In this way, an animal
 may be used to establish a herd or flock of animals having the desired
 genetic characteristic(s).
 Animals produced by transfer of nuclei from a source of genetically
 identical cells share the same nucleus, but are not strictly identical as
 they are derived from different oocytes. The significance of this
 different origin is not clear, but may affect commercial traits. Recent
 analyses of the mitochondrial DNA of dairy cattle in the Iowa State
 University Breeding Herd revealed associated with milk and reproductive
 performance (Freeman & Beitz, In Symposium on Cloning Mammals by Nuclear
 Transplantation (Seidel, G. E. Jr., ed.) 17-20, Colorado State University,
 Colorado (1992)). It remains to be confirmed that similar effects are
 present throughout the cattle population and to consider whether it is
 possible or necessary in specific situations to consider the selection of
 oocytes. In the area of cattle breeding the ability to produce large
 numbers of embryos from donors of high genetic merit may have considerable
 potential value in disseminating genetic improvement through the national
 herd. The scale of application will depend upon the cost of each embryo
 and the proportion of transferred embryos able to develop to term.
 By way of illustration and summary, the following scheme sets out a typical
 process by which transgenic and non-transgenic animals may be prepared.
 The process can be regarded as involving five steps:
 (1). isolation of diploid donor cells;
 (2) optionally, transgenesis, for example by transfection with suitable
 constructs, with or without selectable markers;
 (2a) optionally screen and select for stable integrants--skip for
 micro-injection;
 (3) embryo reconstitution by nuclear transfer;
 (4) culture, in vivo or in vitro, to blastocyst;
 (4a) optionally screen and select for stable integrants--omit if done at
 2a--or other desired characteristics;
 (5) transfer if necessary to final recipient.
 This protocol has a number of advantages over previously published methods
 of nuclear transfer:
 1) The chromatin of the donor nucleus can be exposed to the meiotic
 cytoplasm of the recipient oocyte in the absence of activation for
 appropriate periods of time. This may increase the "reprogramming" of the
 donor nucleus by altering the chromatin structure.
 2) Correct ploidy of the reconstructed embryo is maintained when G0/G1
 nuclei are transferred.
 3) Previous studies have shown that activation responsiveness of
 bovine/ovine oocytes increases with age. One problem which has previously
 been observed is that in unenucleated aged oocytes duplication of the
 meiotic spindle pole bodies occurs and multipolar spindles are observed.
 However, we report that in embryos reconstructed and maintained with high
 MPF levels although nuclear envelope breakdown and chromatin condensation
 occur no organised spindle is observed. The prematurely condensed
 chromosomes remain in a tight bunch, therefore we can take advantage of
 the ageing process and increase the activation response of the
 reconstructed oocyte without adversely affecting the ploidy of the
 reconstructed embryo.
 According to a fourth aspect of the invention, there is provided an animal
 prepared as described above.
 Preferred features of each aspect of the invention are as for each other
 aspect, mutatis mutandis.

The invention will now be described by reference to the accompanying
 Examples which are provided for the purposes of illustration and are not
 to be construed as being limiting on the present invention. In the
 following description, reference is made to the accompanying drawing, in
 which:
 FIG. 1 shows the rate of maturation of bovine oocytes in vitro.
 EXAMPLE 1
 "MAGIC" Procedure using Bovine Oocytes
 Recipient oocytes the subject of this experimental procedure are designated
 MAGIC (Metaphase Arrested G1/G0 Accepting Cytoplast) Recipients.
 The nuclear and cytoplasmic events during in vitro oocyte maturation were
 studied. In addition the roles of fusion and activation in embryos
 reconstructed at different ages were also investigated. The studies have
 shown that oocyte maturation is asynchronous; however, a population of
 matured oocytes can be morphologically selected at 18 hours (FIG. 1).
 Morphological Selection of Occytes
 In FIG. 1 ovaries were obtained from a local abattoir and maintained at
 28-32.degree. C. during transport to the laboratory. Cumulus oocyte
 complexes (COC's) were aspirated from follicles 3-10 mm in diameter using
 a hypodermic needle (1.2 mm internal diameter) and placed into sterile
 plastic universal containers. The universal containers were placed into a
 warmed chamber (35.degree. 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/l
 kanamycin, 30.0 mM Hepes, pH 7.4, osmolarity 280 mOsmols/Kg H.sub.2 O)
 supplemented with 10% bovine serum, transferred into an 85 mm 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 maturation
 medium (TC medium 199 with Earles salts (Gibco), 75 mg/l kanamycin, 30.0
 mM Hepes, 7.69 mM NaHCO.sub.3, pH 7.8, osmolarity 280 mOsmols/Kg H.sub.2
 O) supplemented with 10% bovine serum and 1.times.10.sup.6 granulosa
 cells/ml and cultured on a rocking table at 39.degree. C. in an atmosphere
 of 5% CO.sub.2 in air. Oocytes were removed from the maturation dish and
 wet mounted on ethanol cleaned glass slides under coverslips which were
 attached using a mixture of 5% petroleum jelly 95% wax. Mounted embryos
 were then fixed for 24 hours in freshly prepared methanol: glacial acetic
 acid (3:1), stained with 45% aceto-orcein (Sigma) and examined by phase
 contrast and DIC microscopy using a Nikon Microphot-SA, the graph in FIG.
 1 shows the percentage of oocytes at MII and those with a visible polar
 body.
 Activation of Bovine Follicular Oocytes
 If maturation is then continued until 24 hours these oocytes activate at a
 very low rate (24%) in mannitol containing calcium (Table 1a). However,
 removal of calcium and magnesium from the electropulsing medium prevents
 any activation.
 Table 1a shows activation of bovine follicular oocytes matured in vitro for
 different periods. Oocytes were removed from the maturation medium, washed
 once in activation medium, placed into the activation chamber and given a
 single electrical pulse of 1.25 kV/cm for 80 .mu.s.
 TABLE 1A
 No. of oocytes Hours post onset of Pronuclear formation
 (N) maturation (hpm) [age (hrs)] (% activation)
 73 24 24.6
 99 30 84.8
 55 45 92.7*
 *many 2 or more pronuclei
 Activation Response of Sham Enucleated Bovine Oocytes
 Table 1b shows activation response of in vitro matured bovine oocytes sham
 enucleated at approximately 22 hours post onset of maturation (hpm).
 Oocytes were treated exactly as for enucleation, a small volume of
 cytoplasm was aspirated not containing the metaphase plate. After
 manipulation the oocytes were given a single DC pulse of 1.25 KV/cm and
 returned to the maturation medium, at 30 hpm and 42 hpm groups of oocytes
 were mounted, fixed and stained with aceto-orcein. The results show the
 number of oocytes at each time point from five individual experiments as
 the number of cells having pronuclei with respect to the total number of
 cells.
 TABLE 1B
 No. cells having No. cells having
 pronuclei/Total pronuclei/Total
 no. of cells no. of cells
 EXPERIMENT 30 hpm 42 hpm
 1 1/8 --
 2 0/24 0/30
 3 0/21 0/22
 4 0/27 0/25
 5 0/19 0/1
 hpm = hours post onset of maturation
 Pronuclear Formation in Enucleated Oocytes
 Table 2 shows pronuclear formation in enucleated oocytes fused to primary
 bovine fibroblasts (24 hpm) and subsequently activated (42 hpm). The
 results represent five separate experiments. Oocytes were divided into two
 groups, group A were incubated in nocodazole for 1 hour prior to
 activation and for 6 hours following activation. Group B were not treated
 with nocodazole. Activated oocytes were fixed and stained with
 aceto-orcein 12 hours post activation. The number of pronuclei (PN) in
 each parthenote was then scored under phase contrast. The results are
 expressed as the percentage of activated oocytes containing 1 or more
 pronuclei.
 TABLE 2
 TOTAL 1 PN 2 PN 3 PN 4 PN &gt;4 PN
 GROUP A 52 100 0 0 0 0
 GROUP B 33 45.2 25.8 16.1 3.2 9.7
 The absence of an organised spindle and the absence of a polar body
 suggests that in order to maintain ploidy in the reconstructed embryo then
 only a diploid i.e. G0/G1 nucleus should be transferred into this
 cytoplasmic situation. Incubation of activated oocytes in the presence of
 the microtubule inhibitor nocodazole for 5 hours, 1 hour prior to and
 following the activation stimulus prevents the formation of micronuclei
 (Table 2) and thus when the donor nucleus is in the G0/G1 phase of the
 cell cycle the correct ploidy of the reconstructed embryo is maintained.
 Results
 These results show that:
 i) these oocytes can be enucleated at 18 hours post onset of maturation
 (FIG. 1);
 ii) enucleated oocytes can be fused to donor blastomeres/cells in either
 0.3M mannitol or 0.27M sucrose alternatively the donor the cells or nuclei
 can be injected in calcium free medium in the absence of any activation
 response;
 iii) reconstructed embryos or enucleated pulsed oocytes can be cultured in
 maturation medium and do not undergo spontaneous activation;
 iv) the transferred nucleus is seen to undergo nuclear envelope breakdown
 (NEBD) and chromosome condensation. No organised meiotic/mitotic spindle
 is observed regardless of the cell cycle stage of the transferred nucleus;
 v) such manipulated couplets will activate at 30 hours and 42 hours with a
 frequency equal to unmanipulated control oocytes;
 vi) no polar body is observed following subsequent activation, regardless
 of the cell cycle stage of the transferred nucleus;
 viii) upon subsequent activation 1-5 micronuclei are formed per
 reconstructed zygote (Table 2).
 Reconstruction of Bovine Embryos using "MAGIC" Procedure
 In preliminary experiments this technique has been applied to the
 reconstruction of bovine embryos using primary fibroblasts synchronised in
 the G0 phase of the cell cycle by serum starvation for five days. The
 results are summarised in Table 3.
 Table 3 shows development of bovine embryos reconstructed by nuclear
 transfer of serum starved (G0) bovine primary fibroblasts into enucleated
 unactivated MII oocytes. Embryos were reconstructed at 24 hpm and the
 fused couplets activated at 42 hpm. Fused couplets were incubated in
 nocodazole (5 .mu.g/ml) in M2 medium for 1 hour prior to activation and 5
 hours post activation. Couplets were activated with a single DC pulse of
 1.25 KV/cm for 80 .mu.sec.
 TABLE 3
 NUMBER OF
 BLASTOCYSTS/
 TOTAL NUMBER
 EXPERIMENT OF FUSED %
 NUMBER COUPLETS BLASTOCYSTS
 1 1/30 3.3
 2 4/31 12.9
 EXAMPLE 2
 "MAGIC" Procedure using Ovine Oocytes
 Similar observations to those in Example 1 have also been made in ovine
 oocytes which have been matured in vivo. Freshly ovulated oocytes can be
 retrieved by flushing from the oviducts of superstimulated ewes 24 hours
 after prostaglandin treatment. The use of calcium magnesium free PBS/1.0%
 FCS as a flushing medium prevents oocyte activation. Oocytes can be
 enucleated in calcium free medium and donor cells introduced as above in
 the absence of activation. No organised spindle is observed, multiple
 nuclei are formed upon subsequent activation and this may be suppressed by
 nocodazole treatment.
 Results
 In preliminary experiments in sheep, a single pregnancy has resulted in the
 birth of a single live lamb. The results are summarised in Tables 4 and 5.
 Table 4 shows development of ovine embryos reconstructed by transfer of an
 embryo derived established cell line to unactivated enucleated in vivo
 matured ovine oocytes. Oocytes were obtained from superstimulated Scottish
 blackface ewes, the cell line was established from the embryonic disc of a
 day 9 embryo obtained from a Welsh mountain ewe. Reconstructed embryos
 were cultured in the ligated oviduct of a temporary recipient ewe for 6
 days, recovered and assessed for development.
 TABLE 4
 NUMBER OF
 MORULA, BLA-
 DATE OF STOCYSTS/
 NUCLEAR PASSAGE TOTAL
 TRANSFER NUMBER NUMBER
 17.1.95 6 4/28
 19.1.95 7 1/10
 31.1.95 13 0/2
 2.2.95 13 0/14
 7.2.95 11 1/9
 9.2.95 11 1/2
 14.2.95 12
 16.2.95 13 3/13
 TOTAL 10/78 (12.8%)
 Table 5 shows induction of pregnancy following transfer of all
 morula/blastocyst stage reconstructed embryos to the uterine horn of
 synchronised final recipient blackface ewes. The table shows the total
 number of embryos from each group transferred the frequency of pregnancy
 in terms of ewes and embryos, in the majority of cases 2 embryos were
 transferred to each ewe. A single twin pregnancy was established which
 resulted in the birth of a single live lamb.
 TABLE 5
 PASSAGE
 NUMBER "MAGIC"
 P6 4
 P7 1
 P11 2
 P12 0
 P13 3
 TOTAL MOR/BL 10
 TOTAL NUMBER EWES 6
 PREGNANT EWES % 1 (16.7)
 FOETUSES/ 2/10 (20.0)
 TOTAL
 TRANSFERRED (%)