MXPA98001645A - Oocytes inactivated as receptors of cytopllasto for nuclear transfer - Google Patents

Abstract

A method to reconstitute an animal embryo involves transferring a diploid nucleus to an oocyte, which is detained in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus remains exposed to the recipient cytoplasm for a period. The diploid nucleus can be donated by a cell either in the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. The 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 can then give rise to one or more births of live animals. The invention is useful in the production of transgenic animals, as well as non-transgenic animals, of high genetic merit.

Description

OACYTS INACTIVATED AS RECEPTORS OF CYTOPLLAST FOR NUCLEAR TRANSFER DESCRIPTION OF THE INVENTION This invention relates to the generation of animals, including, but not limited to genetically selected and / or modified animals, and to cells useful in their generation. The reconstruction of mammalian embryos through the transfer of a donor nucleus to an enucleated oocyte or a cell zygote allows the production of genetically identical individuals. This presents clear advantages both for research (ie, as biological controls) and also in commercial applications (ie multiplication of genetically valuable livestock, uniformity in meat products, animal handling).

First, embryo reconstruction through nuclear transfer was proposed (Spemann, Embryonic Development and Induction 210-2111 Hofner Publishing Co., New York (1938)) in order to answer the question of nuclear equivalence or 'if nuclei change during development?'. By transferring nuclei of highly advanced embryonic stages, these experiments were designed to determine at what point the nuclei are restricted in their potential development. 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 can 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 of a single individual can, when transferred to an enucleated egg, give rise to "genetically identical" individuals. In the true sense of the meaning, these individuals could not be clones since the unknown c? Toplasmic contributions in each may vary and also the absence of any chromosomal rearrangements may have been shown. Since the demonstration of embryo cloning in amphibians, similar techniques have been applied to mammalian species. These techniques fall into two categories: 1) transfer from a donor nucleus to a mature metaphase II oocyte, which has had its chromosomal DNA removed and 2) transfer from a donor nucleus to a fertilized cell zygote, which is They have removed both pronuclei. In ungulates, the first procedure became the method of choice since no development was reported using the last one different when the pronuclei are exchanged. The transfer of the donor nucleus to the oocyte cytoplasm is generally achieved by inducing cell fusion. In ungulates, the fusion is induced through the application of a DC electric pulse (direct current) through the contact / fusion plane of the pair. The same pulse, which induces cell fusion also activates the receptor oocyte. After the reconstruction of the embryo, the Further development depends on a large number of factors, including the ability of the nucleus to direct the development, ie, totipotency, development competence of the recipient cytoplasm (ie oocyte maturation), oocyte activation, embryo culture (reviewed by Campbell and Wilmut in Vth World Congress on Genetics as Applied to Livestock 20 180-187 (1994)). In addition to the above, it has been shown that the correct maintenance of the ploidy during the first cycle of the reconstructed embryo cell is of great 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 at a time and only once before mitosis. If either DNA fails replication or is replicated more than once, then the ploidy of that nucleus at the time of mitosis will be incorrect. The mechanisms through which replication is restricted to an individual round during each cell cycle are unclearHowever, several lines of evidence have implied that the maintenance of an intact nuclear membrane is crucial to this control. Morphological cases that occur in the donor nucleus after transfer to an enucleated metaphase II oocyte have been studied in a number of species, including mice (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. Zoo !. 225355-358 (1990)), cow (Kanka et al., Mol. Reprod Dev. 29 110-116 (1991)). Immediately after the fusion the nuclear envelope donor breaks (NEBD), and the chromosomes are prematurely condensed (PCC). These effects are catalyzed through a cytoplasmic activity called maturation promoter factor / mitosis / meiosis (MPF). This activity was found in all the mitotic and meiotic cells that reach a maximum activity in the metaphase. Mature mammalian oocytes are arrested in the metaphase of the 2nd meiotic division (metaphase II) and have high activity MPF. After fertilization or activation, the MPF activity declines, the second meiotic division is completed and the second polar body is extruded, the chromatin is then decondensed and pronuclear formation occurs. In nuclear transfer, reconstructed embryos occur when those of MPF are high in NEBD and PCC; these cases are followed, when MPF activity declines, by decondensation of chromatin and nuclear reformation and subsequent DNA replication. In reconstructed embryos, a correct ploidy can be maintained in one of these two ways; first, by transferring nuclei in a defined cell cycle stage, for example, diploid nuclei of cells in G1, to metaphase II oocytes at the time of activation; or secondly by activating the receiving oocyte and transferring the donor nucleus after the disappearance of the MPF activity. In sheep, this last aspect has produced an increase in the frequency of development to the blastocyst stage from 21% to 55% of reconstructed embryos, when 16-cell embryo blastomeres are used as nuclear donors (Campbell et al., Biol. Reprod. 50 1385-1393 (1994)). These improvements in the frequency of development of reconstructed embryos have not yet been addressed to the issue of nuclear reprogramming. During development, certain genes are "marked", that is, they are altered, so that they are no longer transcribed. Marking studies have shown that this "labeling" is removed during germ cell formation (ie, reprogramming). One possibility is that this reprogramming is affected by the exposure of chromatin to cytoplasmic factors, which are present in cells suffering from meiosis. This increases the question of how this situation can be imitated during the reconstruction of embryos through nuclear transfer, in order to reprogram the development clock of the donor nucleus. It has now been found that nuclear transfer to an oocyte arrested in metaphase II can give rise to a viable embryo, if a normal ploidy (ie, 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 receptor cytoplasm. According to a first aspect of the present invention, a method is provided for reconstructing an animal embryo, the method comprising transferring a diploid nucleus to an oocyte, which is stopped at the metaphase of the second meiotic division are to activate concomitantly the oocyte , keep the core exposed to cytoplasm of the recipient for a period sufficient for the reconstituted embryo to be able to give rise to a live birth and subsequently activate the reconstituted embryo, while maintaining the correct ploidy. At this stage, the reconstituted embryo is an individual cell. In principle, the invention is applicable to all animals, including birds, such as poultry, amphibian species and aquatic species (fish). In practice, however, they will be non-human animals, especially mammals (not humans), particularly placental mammals, where the greatest commercially useful applicability will be observed. This is with ungulates, particularly economically important ungulates such as cattle, sheep, goats, buffalo from India, camels and pigs, where the invention is probably very useful, both as means to clone animals and as means to generate transgenic animals. It should also be noted that the invention is probably also applicable to other economically important animal species such as, for example, horses, llamas or rodents, for example, rats or mice, or rabbits. The invention is equally applicable in the production of transgenic animals, as well as non-transgenic animals. Transgenic animals can be produced from genetically altered donor cells. The whole procedure has a number of advantages over conventional procedures for the production of transgenic (ie, genetically modified) animals, which can be summarized as follows: (1) very few receivers will be required; (2) multiple syngeneic founders can be generated using clonal donor cells; (3) Subtle genetic alteration through the target gene is allowed; (4) all animals produced from embryos prepared by means of the invention must transmit the relevant genetic modification through the germline since each animal is derived from an individual nucleus; in contrast, the production of transgenic animals through pronuclear injection or chimerism after inclusion of stem cell populations modified by blastocyst injection produces a proportion of mosaic animals, in which all cells do not contain the modification and the animal The resultant can not transmit the modification through the germline; and (5) the cells can be selected for the site of genetic modification (eg, integration) prior to generation of the whole animal. It should be noted that the term "transgenic", in relation to animals, should not be taken as limiting with reference to animals that contain in their germ line one or more genes of another species, although many transgenic animals will contain said gene or genes. Rather, the term refers more broadly to any animal whose germline has been the subject of technical intervention by recombinant DNA technology. Thus, for example, an animal in whose germline an endogenous gene has been deleted, duplicated, activated or modified, is a transgenic animal for the purposes of this invention, as well as an animal for whose germline a DNA sequence has been added. exogenous In the embodiments of the invention, where the animai is transgenic, the donor nucleus is genetically modified. The donor nucleus may contain one or more transgenes and the genetic modification may occur before nuclear transfer and reconstitution of the embryo. Although microinjection, analogous to injection to the pronucleus of the male or female of a zygote, can be used as a method of genetic modification, the invention is not limited to that methodology; Transformation and transfection techniques can also be used, for example, electroporation, transfection or viral lipofection. In the method of the invention described above, a diploid core is transferred from a donor to an enucleated receptor oocyte. Donors that are diploid at the time of transfer are necessary in order to maintain the correct ploidy of the reconstituted embryo; therefore, the donors may be either in phase G1 or preferably, as is the subject of our co-pending PCT patent application No. PCT / GB96 / 02099, filed now (claim priority of GB 9517780.4), in the GO phase of the cell cycle. The cycle of the mitotic cell has four distinct phases, G, S, G2 and M. The beginning 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 the beginning. Once a cell has passed through initiation, it passes through the remainder of the G1 phase, which is the pre-DNA synthesis phase. The second stage, the S phase, is when the DNA synthesis is presented. This is followed by the G2 phase, which is the period between DNA synthesis and mitosis. Mitosis itself occurs in phase M. Inactive cells (which include cells in which inactivity has been induced, as well as those cells, which are naturally inactive, such as certain fully differentiated cells) are generally considered non-active. being of none of these four phases of the cycle; they are usually described as being in the GO state, to indicate that they could not normally progress through the cycle. The nuclei of inactive GO cells, such as the nuclei of G1 cells, have a diploid DNA content; both diploid nuclei can be used in the present invention. With respect to the above, it is believed that there is no significant limitation in the cells that can be used in nuclear donors: totally or partially differentiated or undifferentiated cells can be used, since the cells that are cultivated in vitro or abstracted ex vivo. The only limitation is that the donor cells have a normal DNA content and are karyotypically normal. A preferred source of cells is described in co-pending PCT patent application No. PCT / GB95 / 02095, published as WO 96/07732. It is believed that all normal cells contain all 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 the structure of the chromatin, so that the genetic material can redirect the development. The receptor 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 definitively final stage of the egg maturation procedure and then stops. At ovulation, the arrested oocyte is released from the ovary (and, if fertilization occurs, the oocyte is naturally stimulated to complete meiosis). In the practice of the invention, the oocytes can be matured either in vitro or in vivo and are collected at the appearance of the first polar body or as soon as possible after ovulation, respectively. It is preferred that the receiver be enucleated. Since it has generally been assumed that enucleation of receptor oocytes in nuclear transfer procedures is essential, there is no published experimental confirmation of this judgment.

The original procedure described for ungulates involved the division of the cell into two halves, one of which was probably enucleated (Willadsen Nature 320 (6) 63-65 (1986)). This procedure has the disadvantage that another half unknown will still have the metaphase apparatus and that the reduction in cytoplasm volume is thought to accelerate the differentiation pattern of the new embryo (Eviskov et al., Development 109 322-328 (1990) ). More recently, different procedures have been used with the attempt to remove the chromosomes with a minimum of cytoplasm. It was found that aspiration of the first polar body and surrounding cytoplasm removes the metaphase I I apparatus in 67% of sheep oocytes (Smith &Wilmut Bio Reprod 40 1027-1035 (1989)). Only with the use of specific fluorochrome for AD N (Hoechst 33342) was a method provided by which enucleation could 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. Fertile, Sppl 41 125 (1990), Westhusin et al., Biol. Reprod. (Suppl.) 42 176 (1990)). There have been very few reports of non-aggressive aspects for enucleation in mammals, while in amphibians, irradiation with ultraviolet light is used as a routine procedure (Gurdon QJ Microsc. Soc. 101 299-31 1 (1960) ). No reports Detailed information regarding the use of this aspect in mammals, although during the use of DNA-specific fluorochrome, it was observed that the exposure of mouse oocysts to ultraviolet light for more than 30 seconds reduced the development potential of the cell (Tsunoda et al. al., J. Reprod.Fil., 82 173 (1988)). As described above, the enucleation can be achieved physically, by real removal of the nucleus, pro-nuclei metaphase plate (depending on the recipient cell), or functionally, such as by the application of ultraviolet radiation or other enucleation influence. After enucleation, the donor nucleus is introduced either through 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 (that is, in the GO or G1 phase of the cell cycle) at the time of fusion. Once the appropriate donor and recipient cells have been detected, it is necessary that the nucleus of the previous ones be transferred to the latter. More conveniently, the nuclear transfer is effected by fusion. The activation must not be presented at the time of the merger. Three established methods, which have been used to induce fusion are: (1) exposing the cells to chemical compounds that promote fusion, such as polyethylene glycol; (2) use inactivated viruses, such as Sendai virus; and (3) use electrical stimulation. Exposure of cells to fusion-promoting chemical compounds, such as polyethylene glycol or other glycols, is a routine procedure for somatic cell fusion, but has not been widely used with embryos. Since 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 rapidly, may require removal of the zona pellucida (Kanka et al., Mol. Reprod. Dev. 29, 110-116 (1991)). In experiments with mouse embryos, inactivated Sendai virus provides efficient means for cell fusion from embryos of cleavage stage (Graham Wistar Inst. Symp. Monogr. 19 19 (1969)), with the additional experimental advantage of that the activation is not induced. In ungulates, fusion is commonly achieved through the same electrical stimulation that is used to induce partogenetic activation (Willadsen Nature 320 (6) 63-65 (1986), Prather et al., Biol. Reprod. 37 859-866 (1987)). In these species, the Sendai virus induces fusion in a proportion of cases, but is not reliable enough for routine application (Willadsen Nature 320 (6) 63-65 (1986)). Since cell-cell fusion is a preferred method of effecting nuclear transfer, it is not only the 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 carioplasto pair is achieved in the absence of activation by electropulsing in a 0.3M solution of mannitol or a 0.27M solution of sucrose; alternatively, the core can be introduced by injection into 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 the activation of the receptor oocyte. In practice, it is better to enucleate and conduct the transfer as soon as possible after the oocyte reaches metaphase I I. The time this will be after the start of maturation (in vitro) or hormone treatment (in vivo) will depend on the species. For cattle or sheep, the nuclear transfer should preferably be in 24 hours; for pigs in 48 hours; mice, in 12 hours; and rabbits in 20-24 hours. Although the transfer may occur later, it becomes progressively more difficult to achieve as the oocyte ages. High MPF activity is desired. 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! recipient cytoplasm for a sufficient period to allow the reconstructed embryo to be able to, finally, give rise to a live birth (preferably of a fertile offspring). The optimal period before activation varies from species to species and can be easily determined by experimentation. For cattle, a period of 6 to 20 hours is appropriate. The period probably should not be less than that which will allow the formation of the chromosome, and it should not be long enough that the pair spontaneously activates, in extreme cases it dies. When it is the moment of activation, any conventional or other suitable activation protocol can be used. Recent experiments have shown that the requirements for partogenetic activation are more complicated than previously imagined. It has been said that activation is an all-or-nothing phenomenon and that the large number of treatments capable of inducing the formation of a pronucleus all caused "activation". However, the exposure of rabbit oocytes to repeated electrical pulses revealed that only the selection of an appropriate series of pulses and the control of Ca2 + was able to promote the development of diploidized oocytes to middle gestation (Ozil Deveolpment 109 1 17-127 ( 1990)) . During fertilization, there are repeated increases in intracellular calcium concentration (Cutbertson & amp; amp;; Cobbold Natura 31 6 541-542 (1 985)) and it is believed that electrical pulses cause analogous increases in calcium concentration. There is evidence that the pattern of temporary calcium concentrations varies with the species, and You can anticipate that the optimal pattern of electrical pulses will vary in a similar way. 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 increases through exposure to several pulses (Collas &Robi Biol. Reprod. 43 877-884 (1990)). In any individual case, routine adjustments can be made to optimize the number of pulses, the field resistance and the duration of the pulses and the concentration of calcium in the medium. In the practice of the invention, the correct ploidy must be maintained during activation. It is desirable to inhibit or stabilize the microtubule polymerization in order to avoid the production of multiple pronuclei, in order to maintain the correct ploidy. This can be achieved through the application of a microtubule inhibitor such as nocodazole at an effective concentration (such as about 5 μg / ml). Other microtubule inhibitors are colchicine and colcemid. Alternatively, a microtubule stabilizer may be used, such as, for example, taxol.

The molecular component of the microtubules (tubilin) is in a state of dynamic equilibrium between the polymerized and unpolymerized states. Microtubule inhibitors, such as nocodazole, prevent the addition of tubulin molecules to microtubules, thus altering the equilibrium and leading to the depolymerization of the microtubule and the destruction of the spindle. It is preferred to add an inhibitor to the microtubule for a sufficient time before activation to ensure complete, or almost complete, depolymerization of the microtubules. In most cases, it is likely that 30 minutes will be enough. A microtubule stabilizer, such as taxol, prevents spindle breakage and can also prevent the production of multiple pronuclei. The use of a microtubule stabilizer is preferably under conditions similar to those used for microtubule inhibitors. The microtubule inhibitor or stabilizer must remain present after activation until the formation of pronuclei. This must be removed later, and in any case, before the first division is presented. In a preferred embodiment of the invention, at 30-42 hours after the onset of maturation (bovine and ovine, ie 6-18 hours after nuclear transfer), the reconstructed oocytes are placed in a medium containing nocodazole ( 5 μg / ml) and activated using conventional protocols. The incubation in nocodazole can be continued for 4-6 hours after the activation stimulus (depending on the species and age of the oocyte). According to a second aspect of the present invention, a reconstituted, viable animal embryo prepared by a method as previously described is provided. According to a third aspect of the present invention, there is provided a method for preparing an animal, comprising: (a) reconstituting an animal embryo as described above; and (b) having an animal develop from the embryo at term; and (c) optionally, spawning of the animal thus formed. Step (a) has been described in depth previously. The second step, step (b) in the method of this aspect of the invention is to make an animal develop at term from the embryo. This can be done directly or indirectly. In the direct development, the reconstituted embryo from step (a) is simply allowed to develop without further intervention beyond whatever is necessary to allow development to occur. In indirect development, however, the embryo can be further manipulated before development is fully present. For example, the embryo can be divided and the cells clonally expanded, for the purpose of improving performance. Alternatively or additionally, it may be possible that obtain increased yields of viable embryos through the present invention by clonal expansion of donors and / or using the serial (nuclear) transfer method. A limitation in the speed of blastocyst formation currently achieved is due to the fact that the vast majority of embryos do not "reprogram" (although an acceptable number does so). If this is the case, then the speed can be improved as follows. Each embryo that develops on its own can be used as a nuclear donor, such as, for example, in the morula or cell stage 32-64; alternatively, cells of internal cell mass can be used in the blastocyst stage. If these embryos actually reflect those which have reprogrammed gene expression and those nuclei are actually reprogrammed (as seems most likely), then each developing embryo must be multiplied in this way through the efficiency of the transfer procedure nuclear. The degree of improvement likely to be achieved depends on the type of cell. In sheep, it is easily possible to obtain 55% embryos in the blastocyst stage by transferring an individual blastomere from a 16-cell embryo to a preactivated "Universal Receptor" oocyte. So it is reasonable to hypothesize that each embryo developed from an individual cell can give rise to eight in the 16-cell stage. Although these figures are only an approximate guide, it is clear that in the later stages of development, the degree of benefit could depend on the efficiency of the procedure at that stage. In addition to the issuance of conveniences to improve performance, the reconstituted embryo can be cultured, in vivo or in vitro to the blastocyst. Experience suggests that embryos derived by nuclear transfer are different from normal embryos and sometimes benefit from or even require in vivo culture conditions different from those in which embryos are usually cultured (at least in vivo). The reason for this is not known. In the multiplication routine bovine embryos were cultured reconstituted embryos (many of them once) in oviducts of sheep for 5 to 6 days (as described by Willadsen, In Mammalian Egg Transfer (Adams, EE, ed.) 185 CRC Press, Boca Raton, Florida (1982)). In the practice of the present invention, however, in order to protect the embryo, it should preferably be imbibed in a protective medium such as agar before transfer and then dissected from the agar after recovery from the temporary receptor. The function of protective agar or other means is twofold: first, it acts as a structural auxiliary for the embryo, keeping the zona pellucida together; and secondly, it acts as a barrier to cells of the immune system of the recipient animal. Although this aspect increases the proportion of embryos that form blastocysts, there is a disadvantage that a number of embryos may be lost. If in vitro conditions are used, those used routinely in the technique they are totally acceptable. In the blastocyst stage, the embryo can be classified to be suitable for development at term. Typically, this will be done when the embryo is transgenic and the classification and selection of stable members have been made. The classification of non-transgenic genetic markers can also be carried out at this stage. However, since the method of the invention allows the classification of donors at an early stage, which will generally be preferred. After classification, if the classification is performed, the blastocyst embryo is allowed to develop at term. This will generally be in vivo. If the development to blastocyst occurs in vitro, then the transfer to the final recipient animal occurs at this stage. If the blastocyst development has been presented in vivo, although in principle the blastocyst is allowed to develop at term in the pre-blastocyst host, in practice the blastocyst will usually be removed from the pre-blastocyst (temporal) receptor and, after dissection of the protective medium, it will be transferred to the recipient of pre-blastocyst (permanent). In the optional step (c) of this aspect of the invention, animals can be reared from the animal prepared by the preceding steps. In this way, an animal can be used to establish a herd or herd of animals having the desired genetic characteristics.

Animals produced through the transfer of nuclei from a source of genetically identical cells share the same nucleus, but they are not strictly identical since they are derived from different oocytes. The importance of this different origin is not clear, but it can affect commercial aspects. Recent analyzes of mitochondrial DNA from dairy cattle, at Iowa State University Breeding Hard, revealed association with milk yield and reproduction (Freeman &Beitz, in Symposium on Cloning Mammals by Nuclear Transplantation (Seidel, GE Jr., ed.) 17-20, Colorado State University, Colorado (1992)). It is confirmed that similar effects are present throughout the livestock population and consider whether it is possible or necessary in specific situations to consider the selection of oocytes. In the area of livestock breeding, the ability to produce large numbers of embryos from high genetic merit donors can have considerable potential value in disseminating genetic improvement through the national herd. The scale of application will depend on the cost of each embryo and the proportion of transferred embryos capable of developing at term. By way of illustration and summary, the following scheme establishes a typical procedure by which transgenic and non-transgenic animals can be prepared. The procedure can be considered to involve seven steps: (1) isolation of diploid donor cells; (2) optionally, transgenesis, for example, through transfection with suitable constructions, with or without selectable markers; (2a) optionally classifying or selecting stable integrants - jump for micro-injection; (3) reconstitution of the embryo through nuclear transfer; (4) cultivate, in vivo or in vitro, the blastocyst; (4a) optionally classifying and selecting for stable members - omitting if 2a is done - or other desired characteristics; (5) transfer, if necessary, to the final recipient. This protocol has a number of advantages over the previously published methods of nuclear transfer: 1) The chromatin of the donor nucleus can be exposed to the meiotic cytoplasm of the receptor oocyte in the absence of activation during appropriate periods. This can increase the "reprogramming" of the donor nucleus by altering the structure of the chromatin. 2) The correct ploidy of the reconstructed embryo is maintained when the G0 / G 1 nuclei are transferred. 3) Previous studies have shown that the activation sensitivity of bovine / ovine oocytes increases with age. One problem, which has been previously observed, is that in non-enucleated aged crests, the duplication of the meiotic spindle pole bodies occurs and multipolar spindles are observed. Nevertheless , it is reported that in embryos reconstructed and maintained with high levels of MPF, and although the rupture of the nuclear envelope and the chromatin condensation occurred, no organized spindle was observed. The prematurely condensed chromosomes remain in a tight group, therefore, one can take advantage of the aging procedure and increase the activation response of the reconstructed oocyte is adversely affect the ploidy of the reconstructed embryo. According to a fourth aspect of the invention, an animal prepared as described above is provided. Preferred features for each aspect of the invention are as for each aspect to each other, mutatis mutandis. The present invention will now be described with reference to the appended Examples, which are provided for purposes of illustration and are not construed as limiting the present invention. In the following description, reference is made to the accompanying drawing, in which: Figure 1 shows the maturation rate of bovine oocytes in vitro.

EXAMPLE 1"MAGIC" Procedure Using Bovine Oocytes The receptor oocytes, the subject of this experimental procedure, are designated as MAGIC Receptors (Metaphase Arrested G.1 / G0 Acceplng Cytoplast) (Acceptance Cytoplasm G1 / G0 Stopped in the Metaphase). Nuclear and cytoplasmic cases were studied during oocyte maturation in vitro. In addition, fusion and activation roles in embryos reconstructed at different ages were investigated. Studies showed that oocyte maturation is asynchronous; however, a population of mature oocytes can be morphologically selected at 18 hours (Figure 1).

Morphological Selection of Oocytes In Figure 1, ovaries were obtained from a local cattle slaughterhouse and maintained at 28-32 ° C during transportation to the laboratory. The cumulus oocyte complexes (COC) of follicles with a diameter of 3-10 mm were aspirated using a hypodermic needle (1.2 mm internal diameter) and placed in universal, sterile plastic containers. The universal containers were placed in a warm chamber (35 ° C) and the follicular material was allowed to settle for 10-15 minutes before emptying 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 nOsmoles / Kg H 0) supplemented with 10% bovine serum, they were transferred to an 85 mm Petri dish and the COCs were searched under a dissection microscope. The complexes were selected with at least 2-3 layers compact cells were washed three times in the dissecting medium and transferred to the maturation medium (TC 199 medium with Eagles salts (Gibco), 75 mg / l kanamycin, 30.0 mM Hepes, 7.69 mM NaHCO3, pH 7.8, osmolarity of 280 nOsmoles / Kg of H2O) supplemented with 10% of bovine serum and 1x106 of granulosa cells / ml and were cultivated until required in an oscillating table at 39 ° C in an atmosphere of 5% C02 in air. The oocytes were removed from the maturation box and moistened mounted on glass slides cleaned with ethanol under coverslips, which were joined using a mixture of 5% petroleum jelly and 95% wax. The assembled embryos were then fixed for 24 hours in freshly prepared methanol: glacial acetic acid (3: 1), stained with 45% aceto-orcein (Sigma) and examined through phase contrast and DIC microscope using Nikon Micropht -SA, the graph in Figure 1 shows the percentage of oocytes in the Mil and those with a visible polar body.

Activation of Folicular Bovine Oocytes If maturation is then continued up to 24 hours, these oocytes are activated at a very low rate (24%) in mannitol containing calcium (Table 1a). However, the removal of calcium and magnesium from the electropulsing medium avoids any activation.

Table 1a shows the activation of bovine follicular oocytes matured in vitro during different periods. The oocytes they were removed from the maturation medium, washed once in activation medium, placed in the activation chamber and applied a single electric pulse of 1.25 kV / cm for 80 μs.

TABLE 1a 2 or many more pronuclei Response of Activated Enucleated Bovine Oocytes in Copy Table 1b shows the activation response of a copy of enucleated in vitro matured bovine oocytes at approximately 22 hours after the onset of maturation (hpm). The oocytes were treated exactly as for enucleation, a small volume of cytoplasm was aspirated without containing the metaphase plate. After manipulation, a single DC (direct current) pulse of 1.25 KV / cm was applied to the oocytes and they were returned to the ripening 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 in each point of time from five experiments as the number of cells that have pronuclei with respect to the total number of cells.

TABLE 1 b hpm = hours after the start of maturation Pronuclear Formation in Enucleated Occitos Table 2 shows pronuclear formation in elucidated oocytes fused to primary bovine fibroblasts (24 h pm) and subsequently activated (42 hpm). The results represent n five experiments separately. The oocytes were divided into two groups, group A were incubated in nocodazole for 1 hour before activation and for 6 hours after activation. Group B was not treated with nocodazole. Activated oocytes were fixed and stained with aceto-orcein 12 hours after activation. The number of pronuclei (PN) in each partner after it was classified under phase contrast. The results are expressed as the percentage of activated oocytes containing 1 or more pronuclei.

TABLE 2 The absence of an organized spindle and the absence of a polar body suggests that in order to maintain the ploidy in the reconstructed embryo then only one diploid, ie, the G0 / G1 core, must be transferred to this cytoplasmic situation. The incubation of activated oocytes in the presence of the microtubule inhibitor, nocodazole, for 5 hours, 1 hour before and after 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: i) these oocytes can be enucleated at 18 hours after the start of maturation (Figure 1); ii) the enucleated oocytes can be fused to the blastomeres / donor cells, either 0.3 M mannitol or 0.27 M of sucrose, alternatively the donor, the cells or the nuclei can be injected into the calcium-free medium in the absence of any activation response. iii) reconstructed embryos or enucleated pulsed oocytes can be cultured in a maturation medium and not undergo spontaneous activation; iv) the transferred core is seen to undergo rupture of the nuclear envelope (N EBD) and condensation of the chromosome. No organized meiotic / mitotic spindle was observed, without considering the cycle stage of the transferred core cell; v) said manipulated pairs will be activated in 30 hours and 42 hours with a frequency equal to the control oocytes manipulated; vi) no polar body was observed after subsequent activation, without considering the cycle stage of the transferred core cell; viii) after activation, 1 -5 micronuclei were formed per reconstructed zygote (Table 2).

Reconstruction of Bovine Embryos Using the "MAGIC" Procedure In preliminary experiments, this technique has been applied to the reconstruction of bovine embryos using primary fibroblasts in the GO phase of the cell cycle through starvation. serum for five days. The results are summarized in Table 3. Table 3 shows the development of bovine embryos reconstructed through nuclear transfer of primary bovine fibroblasts (GO) with starvation of inactivated, enucleated Mi ocyte serum. The embryos were reconstructed at 24 hpm and the fused pairs were activated at 42 hpm. The fused pairs were incubated in nocodazole (5 μg / ml) in the M2 medium for 1 hour before activation and 5 hours after activation. The pairs were activated with a single DC pulse of 1.25 KV / cm for 80 μsec.

TABLE 3 EXAMPLE 2"MAGIC" Procedure Using Ovine Oocytes Similar observations were also made to those of Example 1 in sheep oocytes, which were killed in vivo. Newly ovulated oocytes could be recovered by washing from the oviducts of superestimulated female sheep, 24 hours after Prostaglandin treatment The use of calcium-magnesium-free PBS / 1.0% FCS as a washing medium prevents oocyte activation. The oocytes can be enucleated in a calcium-free media and donor cells were introduced, as was done previously, in the absence of activation. No organized spindle was observed, multiple nuclei were formed after activation and this can be suppressed through nocodazole treatment.

Results: In preliminary experiments with sheep, an individual pregnancy resulted in the birth of a single live lamb. The results are summarized in Tables 4 and 5. Table 4 shows the development of reconstructed sheep embryos through the transfer of an established embryo-derived cell line to ovine matured in vivo, enucleated, inactivated oocytes. The oocytes were obtained from supersimulated Scottisk Blackface female sheep, the cell line was established from the embryonic disc of a 9 day old embryo of a female Weish Mountain sheep. The reconstructed embryos were cultured in the bound oviduct of a temporary female recipient sheep for 6 days, recovered and analyzed for development.

TABLE 4 Table 5 shows the induction of pregnancy after the transfer of all the reconstructed embryos from the morula / blastocyst stage to the uterine horn of final recipient Blackface ewes. The Table shows the total number of embryos of each group transferred to the pregnancy frequency in terms of females and embryos, in most cases 2 embryos were transferred to each female. A single double pregnancy was established, which resulted in the birth of a single living lamb.

TABLE 5

Claims (8)

1. - A method for reconstructing an animal embryo, the procedure comprising transferring a diploid nucleus to an oocyte, which is stopped at the metaphase of the second meiotic division without concomitantly activating the oocyte, keeping the nucleus exposed to the cytoplasm of the receptor for a period enough for the embryo to be able to give rise to a live birth and subsequently activate the reconstituted embryo while maintaining a correct ploidy.

2. A method according to claim 1, wherein the animal is a species of ungulate.

3. A method according to claim 2, wherein the animal is a cow or bull, pig, goat, sheep, camel or buffalo from India.

4. A method according to any of claims 1 to 3, wherein the donor nucleus is genetically modified.

5. A method according to any of claims 1 to 4, wherein the diploid core is donated by an inactive cell.

6. A method according to any of claims 1 to 5, wherein the oocyte is enucleated. 7 - A method according to any of claims 1 to 6, wherein the nuclear transfer is achieved at through the fusion of the cell. 8. A method according to any of claims 1 to 7, wherein the animal is a cow or bull, and wherein the donor nucleus is kept exposed to the recipient cytoplasm for a period of 6 to 20 hours before activation. . 9. A method according to any of claims 1 to 8, wherein the correct ploidy is maintained during activation through the inhibition of the microtubule. 10. A method according to claim 9, wherein the inhibition of the microtubule is achieved through the application of nocodazole. 11. A method according to any of claims 1 to 8, wherein the correct ploidy is maintained during activation through the stabilization of the microtubule. 12. A method according to claim 11, wherein the stabilization of the microtubule is achieved through the application of taxol. 13. A method for preparing an animal, the method comprising: (a) reconstituting an animal embryo as described in the preceding claim; and (b) having an animal develop from the embryo at term; and (c) optionally, spawning of the animal thus formed. 14. A method according to claim 13, wherein the animal embryo is also manipulated before the embryo's full development. 15. A method according to claim 14, wherein more than one animal is derived from the embryo. 16. A reconstituted animal embryo, which is capable of giving rise to a live birth and is prepared through a method according to any of claims 1 to 12. 1

7. An animal prepared through the method of according to any of claims 13 to 15. 1

8. An animal developed from an embryo according to claim 16.