EP1465994A2

EP1465994A2 - Rabbit nuclear cloning method and uses thereof

Info

Publication number: EP1465994A2
Application number: EP03718816A
Authority: EP
Inventors: Patrick Chesne; Pierre Adenot; Jean-Paul Renard
Original assignee: Institut National de la Recherche Agronomique INRA
Current assignee: Institut National de la Recherche Agronomique INRA
Priority date: 2002-01-10
Filing date: 2003-01-10
Publication date: 2004-10-13
Also published as: WO2003059053A3; AU2003222862A1; AU2003222862A8; CA2472452A1; FR2834518A1; WO2003059053A2; JP2005525098A; FR2834518B1; US20050155093A1; US20100293623A1

Abstract

The invention concerns a method for producing non-human mammal embryos, in particular rabbit by nuclear cloning. The invention also concerns the mammals obtained and their uses.

Description

Nuclear cloning process in rabbits and uses

The present invention relates to a process for nuclear cloning of vertebrates, in particular mammals, and more particularly rabbits. 1 / invention also relates to the animals thus produced, in particular rabbits, at the fetal or adult stage, as well as to their use for the production of molecules of interest or as animal models for studying human pathologies.

The rabbit is increasingly considered in the biotechnology industry for the advantages it provides compared to other animal species. First of all, from a phylogenic point of view, the rabbit is closer to primates, that is to say to humans, than rodents, mice and rats, commonly used today (Graur et al., 1996). Then, the rabbit is better suited to physiological manipulations due to its size, unlike small animals such as rodents, mice and rats, or large animals, such as cows, goats, sheep, pigs, etc. Finally, the large litter size and rapid reproduction are all assets for a laboratory animal intended for the study of human pathologies or for the production of recombinant proteins of interest. For example, the rabbit model is of great interest for the development of a clinical treatment for arteriosclerosis (Hoeg et al., 1996) and cystic fibrosis (Chen et al., 2001). The nuclear atomic transfer associated with genetic modifications of the donor cells of nuclei could make extremely interesting the use of the rabbit as laboratory animal (Fan et al., 1999) which for the moment is confined to the production of recombinant proteins d interest in large quantities (Stinnackre et al., 1997). Nuclear transfer technology is very interesting because it allows the rapid production of a large number of genetically identical animals, or their descendants, with particular genetic characteristics. However, to date, nuclear transfer in rabbits has never been successfully implemented (Yin et al., 2000; Dinnyés et al., 2001) despite the pioneering role of rabbits in testing experiments. point of nuclear cloning technology (Bromhall et al., 1975).

Only one research laboratory was able to obtain the beginnings of embryo pregnancies from somatic cell nuclei (Yin et al., 2000), but surprisingly none of these gestations was able to reach their term.

The rabbit, although initially used as an animal model for the development of nuclear transfer techniques (Bromhall et al., 1975) therefore appears to be a species for which existing nuclear cloning technologies, which have given success with others species such as sheep (ilmut et al., 1997; WO 97 07669), mice (Wakayama et al., 1998; WO 99 37143), cattle (Wells et al., 1999), goats (Baguisi et al. , 1999; WO 00 25578), pork (Polejaeva et al., 2000), are not suitable. he There is therefore a great need to develop a new process for nuclear cloning of animal species which hitherto cannot be cloned efficiently by the nuclear transfer methods currently available, and in particular to develop a process for nuclear cloning in rabbits, which is both efficient, reproducible and which makes it possible to obtain good cloning yield.

This is the problem which the present invention proposes to solve by providing a process for the production of mammalian embryos comprising the following steps:

(a) evaluate and / or determine the development asynchrony (T) between two embryos of the same species and of the same age: the first embryo being produced by crossing at time t ₀ of a male preferably vasecto ised with a female having preferably received hormone therapy to increase ovulation, said first embryo being at least cultured and / or manipulated in vitro; the second embryo being produced by crossing at time to of a fertile male with a female having preferably received hormonal treatment to increase ovulation, the said second embryo being normally fertilized or obtained by parthenogenetic activation, the evaluation and / or determination taking place at the latest on the day of uterine implantation of said second embryo and;

(b) transferring an embryo at least cultivated and / or manipulated in vi tro in the uterus of a recipient female having been crossed with a vasectomized male at time t = t ₀ + T (+/- 25% T);

(c) optionally, allow the said embryo transferred to step b) to implant and develop in the uterus of the said recipient female.

In principle, the present invention is applicable to all mammals. More particularly, the animal according to the invention is a mammal. The invention is particularly interesting for mammals such as ungulates, equines, camelids, rodents, lagomorphs and primates. Among the rodents, it is worth mentioning the mouse, the rat, the hamster, the guinea pig. Among the ungulates, it is worth mentioning cattle, sheep, goats, pigs. Preferably, the mammal according to the invention is a lagomorph. The invention is particularly interesting for animals which until now were difficult to obtain or even impossible to obtain by nuclear cloning, such as the rabbit or the rat. By “asynchrony”, within the meaning of the present invention, is meant the time or delay expressed in hours which exists at a given instant in embryonic development between the stage of development of a normally fertilized embryo and which develops according to the laws of the nature and stage of development of an embryo which at some point in its development has at least been manipulated in vi tro, two embryos being of the same age and the same species. By embryos of the same age is meant to define that the embryos were conceived simultaneously or at the same time. Thus, in the case of a normally fertilized embryo and a reconstituted embryo obtained by nuclear transfer, the enucleated oocyte will have the same age as the oocyte normally fertilized by a spermatozoon. The two embryos can belong to the same animal species but also to different species. In the case of nuclear cloning in rabbits, the two embryos are rabbit embryos. These two embryos may or may not be of different races such as the “Ne-Zealand”, “Fauve-de-Bourgogne”, “Argenté-de-Champagne”, Californiennes, “Géant-de-Bouscat” races, or all races whose zoological specificities are defined in an official standard (The rabbit breed, Ed. 2000 French Federation of Cuniculture (Ed.) or derived from crosses having given rise to commercial strains of rabbits such as the strain GD22 / 1077. By "cultivated in vitro ”Is understood to mean, for the purposes of the present invention, an embryo which is not naturally conceived and / or developed, that is to say an embryo for which at least one stage of its conception and / or of its development is carried out in vitro For example, the embryo "cultivated in vi tro" within the meaning of the present invention is cultivated and develops in an appropriate culture medium containing the nutrients necessary for the growth and / or the differentiation of the 'embryo n. "Manipulated in vitro" means, in the sense of the present invention, an embryo cultivated in vitro obtained by nuclear transfer and / or modified genetically by trangenesis. The embryo is cultured and / or handled in vitro at the latest until the day before implantation.

According to the present invention, the evaluation and / or determination of asynchrony is carried out at the latest on the day of uterine implantation of the embryo normally fertilized or obtained by parthenogenic activation or by cloning; however, this determination of asynchrony is preferably carried out at a development stage chosen from the 1 cell stage, the 2 cell stage, the 4 cell stage, the 8 cell stage, the 16 cell stage, the morula stage and the stage blastocyst. Preferably, the evaluation and / or determination of 1 asynchrony is carried out from embryo (s) having reached the blastocyst stage in vitro and whose development kinetics is compared to that of embryos obtained in vivo.

This evaluation and / or determination of the developmental asynchrony T is preferably carried out by cell counting or determination of the proportion of embryonic cells organized into internal cell mass, cells which will contribute to the formation of the fetus and of a part of the placenta. However, other technologies known to those skilled in the art can be used to carry out this evaluation and / or determination, such as for example the demonstration of expression and / or lack of expression. cell markers characteristic of a particular stage of embryonic development.

The development asynchrony T is preferably greater than or equal to 15 hours, preferably greater than or equal to 4 p.m., greater than or equal to 5 p.m., greater than or equal to 6 p.m., greater than or equal to 7 p.m., greater than or equal to 8 p.m., greater than or equal to 9 p.m., greater than or equal to 10 p.m., greater than or equal to 11 p.m. , greater than or equal to 24:00, greater than or equal to 25:00, greater than or equal to 26:00, greater than or equal to 27:00, greater than or equal to 28:00, greater than or equal to 29:00, or greater than or equal to 30:00. More preferably, said development asynchrony T is approximately 24 hours.

In the method according to the present invention, said embryo transferred to step b) is cultivated under the same conditions as said first embryo. According to a first preferred embodiment, the said embryo transferred in step b) is in the 1 cell stage. According to a second embodiment, said embryo transferred in step b) is in the 2 cell stage. According to a third embodiment, the said embryo transferred in step b) is in the 4 cell stage. Alternatively, the said embryo transferred to step b) is at the 8 cell stage, at the 16 cell stage, at the 32 cell stage, at the 64 cell stage, or at a more advanced stage of development.

Methods of obtaining vasectomized male animals are well known to those skilled in the art, as well as hormonal treatments intended to increase ovulation (see for example: Kennely and Foote, 1965).

Said embryo transferred to step b) of the method according to the invention develops into a fetus, preferably said fetus develops into a newborn, and said newborn develops into an adult. It is therefore It is also an object of the present invention to provide an embryo, fetus, newborn baby, adult mammal, except man, or cells derived therefrom, produced by a method comprising or including the method according to the invention. 'invention. The invention also relates to the progeny of said adult mammal according to the invention. For ethical reasons, it is obvious that the methods according to the invention must not be implemented for the purpose of reproductive cloning of human beings.

Depending on the needs, it may be advantageous to stop the development or gestation of the embryo at the embryonic or fetal stage to derive cells, in particular cells from the internal cell mass, such as stem cells from said embryo. The term “embryonic stem cells” is intended to denote the undifferentiated pluripotent cells, which can be cultivated in vi tro for a long time without losing their characteristics, and which are capable of differentiating into one or more cell types when they are placed in defined culture. Thus, when the stem cells according to the invention are ES cells, it is possible to envisage inducing the differentiation of these into different cell types such as for example muscle, cardiac, glial, nervous, epithelial, hepatic, pulmonary cells, pancreatic. Thus, in the context of so-called "therapeutic" cloning, the embryo can be a human embryo obtained by a nuclear cloning process, intra- or interspecies, in order to obtain useful stem cells, differentiated or not for preventive or curative treatment of patients requiring such treatment. In this case, steps b) of transferring the embryo into the uterus and c) of implanting the transferred embryo, of the method according to the invention, are optional. Those skilled in the art know the techniques for in vitro culture of cells of the internal cell mass (see for example WO 97 37009) and embryonic stem cells in particular, so that these retain their totipotency or their pluripotency in culture ( Evans et al., 1981; EP 380 646; WO 97 30151) or so that these induce their differentiation in a particular cell type.

Stage b) of the process according to the invention relates to the development of the non-human animal from the embryonic stage to its term. This can be done directly or indirectly. In a direct development, the reimplanted embryo, transgenic or not, reconstituted or not, is simply left to develop in the uterus of the carrier female without any foreign intervention occurring until the end. In indirect development, the embryo can be manipulated before full development is achieved. For example, the embryo can be divided, and cells grown clonally, in order to increase the production yield of cloned animals. Alternatively or additionally, it is possible to increase the production yield of viable embryos by the successive implementation of the nuclear transfer method according to the invention.

The embryo, fetus, newborn, adult mammal, except man, or cells derived therefrom, obtained by the implementation of the method according to the invention can also be transgenic. Transgenesis is either carried out during the in vitro culture of the embryo, or that the embryo is derived from an animal itself transgenic. For the purposes of the present invention, the term “transgenic” is intended to denote a cell or an animal comprising at least one transgene. The term “transgene” is intended to denote genetic material which has been or which is going to be inserted artificially into the genome of a cell of an animal according to the invention, particularly in a mammalian cell cultivated in vitro or in a cell living mammal and which will remain there in the said cell in episomal form. The methods for generating transgenic cells according to the invention are well known to those skilled in the art. They include, but are not limited to, the technology for targeted inactivation of one or more genes by homologous recombination ("Knock-Out"), the technology for targeted insertion of one or more genes by homologous recombination ("Knock-In" ”), The technology of random integration of a transgene by micro-injection into the nucleus. The transgene according to the invention, optionally included in a linearized vector or not, or in the form of a vector fragment, can be introduced into the host cell by standard methods such as for example microinjection into the nucleus (US 4 873 191), transfection by precipitation with calcium phosphate, lipofection, electroporation, thermal shock, transformation with cationic polymers (PEG, polybrene, DEAE-Dextran ...), viral infection, sperm.

The reimplantation of the embryo into the uterus of a carrier female uses techniques known to those skilled in the art. Usually, the surrogate mother is anesthetized and the embryos are inserted into the oviduct. The number of embryos implanted in a particular host varies according to the species but is usually compatible with the number of newborns usually produced by said species. The transgenic descendants or not of the surrogate mother are screened for the presence and / or the expression of the transgene or of a marker characteristic of the said descendants using the appropriate methods. The screening is often carried out by Southern blotting or by northern blot analysis using a probe which is complementary to at least a portion of the transgene or of the marker. A Western blot analysis using an antibody against the protein encoded by the transgene or said marker can be used as an alternative or as an additional method to screen for the presence of the protein product encoded by the transgene or said marker. Typically, DNA is prepared from animal cells, and in particular from lymphocytes in rabbits, then analyzed by southern blotting or by PCR for the presence of the transgene. Alternatively, the tissue of the cells capable of expressing the transgene or the marker with the highest level are tested for the presence and / or the expression of the transgene or of the marker using the analysis by Southern blot or by PCR. Alternatively, additional methods to assess the presence of the transgene or the marker are biochemical methods, such as enzymatic and / or immunological tests, histological methods to detect the presence of a particular marker or certain enzymatic activities, analyzes by flow cytometry.

It is also one of the objects of the present invention to provide an in vitro method for cloning mammals by nuclear transfer comprising or including a method according to the invention for producing embryos of non-human mammals which comprises at least step d evaluation and / or determination of the developmental asynchrony T between two embryos of the same species and of the same age. The term “nuclear transfer or nucleus transfer” is intended to denote the transfer of nucleus from a donor cell originating from an animal according to the invention, preferably from a mammal, at a stage of development comprised between the embryonic to adult stage, in the cytoplasm of an enucleated recipient cell of the same or a different species. In general, the recipient cell is an oocyte. The transferred nucleus is reprogrammed to direct the development of cloned embryos which can then be transferred into the uterus of surrogate females to produce fetuses and newborns, or used to produce cells from the cultured internal cell mass.

The donor genetic material is introduced by various means into the enucleated recipient cell to form the reconstituted embryo. Generally the donor genetic material is introduced by fusion using methods such as (i) exposure of cells to chemical agents promoting fusion such as ethanol, polyethylene glycol (PEG) or other glycols; (ii) the use of biochemical agents, such as phytohaemagglutinin (PHA); (iii) the use of inactivated viruses, such as the Sendai virus; (iv) the use of liposomes; (v) the use of electro-fusion. The present invention is not limited to the use of these fusion techniques and although cell-cell fusion is the preferred method for carrying out nuclear transfer McGrath and Solter, 1984; WO 99 37143), other equally preferred methods can be implemented, such as micro-injection, preferably micro-injection of the donor nucleus (Wakayama et al., 1998). According to the present invention, the donor cell and the recipient cell, preferably an oocyte, come from the same animal, or from two animals of the same species. According to another embodiment, the donor cell and the recipient cell come from two animals of different species.

The combination of the genome of the activated donor cell and the activated oocyte produces the embryo obtained by nuclear transfer, or NT embryo (for “nuclear transfer”), also called reconstituted embryo, terms which will be used interchangeably in the present patent.

The donor cell according to the invention can be any cell type which contains a genome or genetic material, such as somatic cells, germ cells, embryonic cells such as pluripotent stem cells, totipotent stem cells, such as embryonic stem cells for example (ES cells). The term "somatic cell" refers to differentiated diploid cells. The somatic cell can either come from an animal, or from a culture of cells or tissues which have undergone at least one passage in culture and which have been frozen or not. When the somatic cell is derived from an animal, the animal can be at any stage of development, for example an embryo, a fetus or an adult. Somatic cells preferably include and are not limited to fibroblasts (eg, primary fibroblasts), epithelial cells, muscle cells, cumulus cells, neural cells, mammary cells, hepatocytes, Langerhans cells. Preferably, the somatic donor cells are cumulus cells. Somatic cells can be obtained for example by dissociation of tissues by mechanical or enzymatic means (in general by the use of trypsin or proteases) to obtain a cell suspension which is in general cultured until obtaining a confluent cell monolayer. Somatic cells can be harvested or prepared for cryopreservation and kept frozen until further use. The nucleus donor cells are either proliferative or quiescent. The state of quiescence which corresponds to the Go / Gl stage of the cell cycle is obtained in cultured cells by inhibition of contact or by deprivation of serum (Whitfield et al., 1985). The proliferative state can be considered as corresponding to all other stages of the cell cycle.

The recipient cells according to the present invention are preferably oocytes, more preferably activated oocytes. Activated oocytes are those which are in a stage of meiotic cell division which includes prophase, anaphase, metaphase, telophase I and II, preferably metaphase I, anaphase I, anaphase II, and preferably telophase II. The invention also relates to metaphase II oocytes which are considered to be in a state of rest, but which can be activated by techniques known to those skilled in the art (WO 00 25578). The state of development of the oocyte is defined by a visual inspection of the oocyte under sufficient magnification. Oocytes which are in telophase II are for example identified by the presence of a protusion of the plasma membrane corresponding to the second polar globule. Methods for identifying the different stages of meiotic cell division are known to those of skill in the art.

Various techniques have been described for activating oocytes, such as the use of calcium ionophores (for example ionomycin) (see US Pat. No. 5,496,720) which are agents which increase the permeability of the membrane of oocytes and allow the calcium from entering the oocytes. Also ethanol which has the same effects can be used. Also the activation of oocytes can be achieved by electrical stimulation trains which can be used in rabbits to control the calcium levels in the oocyte (Ozil and Huneau, 2001). Preferably, the state of activation of the oocyte is obtained by a train of electric pulses then chemically prolonged by culturing the oocytes in the presence of protein kinase inhibitor such as 6-dimethyl-amino-purine (6-DMAP ) and / or in the presence of an inhibitor of peptide synthesis such as cycloheximide (CHX). The step of activating the oocyte can be carried out before, during and / or after the step of fusing the nucleus or the donor cell with the recipient oocyte.

Oocytes can be obtained by in vitro maturation of material obtained by follicular puncture of ovaries collected in slaughterhouses or by aspiration of oocytes from the follicles of the ovaries at specific times in the reproductive cycle of a female who has been hormone-stimulated or not. exogenously (female super-ovulated). Oocytes are mature in vivo or in vitro up to metaphase II or telophase stage. All mature oocytes in vivo must be harvested by washing the oviduct in PBS (phosphate buffered saline) buffer. The mature oocytes in vitro are collected and transferred to a culture medium containing 10% of serum, such as fetal calf serum (FCS, "fetal calf serum"). The oocytes are stripped of the cumulus cells and then enucleated as previously described by Adenot et al. (1997) .

The present invention relates more particularly to a method for producing rabbit embryos comprising the following steps: a) evaluate and / or determine the development asynchrony (T) between two rabbit embryos of the same age: the first embryo being produced by crossing at time trj of a male preferably vasectomized with a female having preferably received a treatment hormonal to increase ovulation, said first embryo being at least cultured and / or manipulated in vitro; the second embryo being produced by crossing at time trj of a fertile male with a female having preferably received a hormonal treatment to increase ovulation, the second embryo being normally fertilized or obtained by parthenogenetic activation; the evaluation and / or determination taking place at the latest on the day of uterine implantation of said second embryo normally fertilized or obtained by parthenogenetic activation; and b) transfer a rabbit embryo cultivated and / or manipulated in vitro, not older than the blastocyst stage into the uterus of a recipient female having been crossed with a vasectomized male at time t = t ₀ + T (+ / -25% T); c) optionally, leave the said embryo transferred in step b) to implant and develop in the uterus of the said recipient female. The evaluation and / or determination is carried out at a stage of development between days Dl and D10 post coitum, preferably between days Dl and D8 post coitum. More preferably, the evaluation and / or the determination is carried out in vitro on days D3 and D4 post coitum. In the method for producing rabbit embryos according to the invention, the so-called developmental asynchrony T is 23 hours +/- 25%. Preferably, said asynchrony is approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours or approximately 24 hours.

According to a preferred embodiment of the invention, the said rabbit embryo cultivated and / or manipulated in vi tro is a transgenic embryo. According to another preferred embodiment, said embryo cultured and / or manipulated in vitro is a reconstituted embryo obtained by nuclear transfer. More preferably, said embryo cultivated and / or manipulated in vitro is a reconstituted transgenic embryo obtained by nuclear transfer. In the method for producing rabbit embryos according to the present invention, said embryo transferred in step b) is preferably at the 1, 2 or 4 cell stage, although later stages of development can be envisaged. Rabbit and / or fetal embryos, newborns, adult rabbits, descendants of adult rabbits, or cells derived therefrom, produced by a process comprising or including the method of producing rabbit embryos according to the invention are also the object of the present invention.

The invention also relates to an in vitro method of rabbit cloning by nuclear transfer comprising or including a method of producing rabbit embryos, preferably transgenic, according to the invention. More particularly, this in vitro method of rabbit cloning by nuclear transfer comprises the steps of: a) insertion of a rabbit donor cell or of a rabbit donor cell nucleus into an enucleated rabbit oocyte under conditions allowing obtain a reconstituted embryo; b) activation of the reconstituted embryo obtained in step a); c) transfer of said reconstituted embryo to a carrier rabbit, so that the reconstituted embryo develops into a fetus, and possibly into a newborn; and is characterized in that the method comprises or includes a method of producing rabbit embryos according to the invention. Preferably, the transfer of nucleus into the recipient cytoplasm is carried out by fusion of the donor cell and the recipient cytoplasm; alternatively, the transfer of nucleus into the recipient cytoplasm is carried out by micro-injection of the donor nucleus into the recipient cytoplasm.

In order to successfully carry out nuclear cloning in animal species hitherto refractory to such a technology such as the rabbit and the rat, or in animal species which are difficult to clone, it is also important that the in vitro activation phase of the reconstituted embryo is as brief as possible. Indeed, in species such as rabbits, this phase S occurs very quickly compared to other species (Szôllôsi, 1966). The shortening of the Activation procedure allows the DNA replication phase (phase S) of the first cell cycle to occur, relative to ovulation, at a time identical to that of normal development. The present invention therefore provides means of reducing the in vitro activation phase of the reconstituted embryo, in order to guarantee sufficient development kinetics so that the embryo does not end up outside the implantation window even after desynchronization. . The inventors have thus demonstrated, quite surprisingly, that the mixture of two drugs, one being an inhibitor of protein kinase, such as 6-dimethylaminopurine (6-DMAP), and at least one inhibitor of synthesis protein, such as cycloheximide (CHX), hitherto used separately to carry out the activation of reconstituted embryos, made it possible to obtain more effective activation over shorter times while limiting the known side effects of these drugs, in particular on initiation of phase S and DNA replication. Also, in the method according to the invention, the activation phase during the in vitro culture is carried out by adding preferably simultaneously, or successively or offset in time, to the culture medium of said reconstituted embryo, at least at least one protein kinase inhibitor and at least one protein synthesis inhibitor. Preferably, said activation is carried out by simultaneous addition of 6-DMAP and cycloheximide (CHX). The concentrations of 6-DMAP and CHX to achieve such activation in rabbits are respectively between 1 and 5 millimolar (mM), preferably 2 mM and 1 to 10 micrograms (μg) preferably 5μg per ml. The duration of activation preferably ranges from 30 minutes to 2 hours, preferably one hour. Those skilled in the art will easily adapt these parameters accordingly for other mammals than the rabbit.

The present invention therefore also relates to a method for activating a reconstituted embryo or not, transgenic or not, characterized in that it comprises the step of adding to the culture medium of said embryo, successively, simultaneously, or time-shifted, at least one protein kinase inhibitor, more particularly involved in the recovery of meiosis, and preferably 6-DMAP, and at least one inhibitor of protein synthesis, preferably cycloheximide (CHX).

The present invention also relates to an in vitro mammalian cloning method as described above, comprising the steps: (i) of insertion of a donor cell or of a donor cell nucleus into an enucleated mammalian oocyte of the same species or a species different from that of the donor cell, under conditions allowing to obtain a reconstituted embryo; (ii) activating the reconstituted embryo obtained in step (i); and (iii) transfer of said reconstituted embryo into a female carrying a mammal, so that the reconstituted embryo develops into a fetus, characterized in that said activation is carried out by successive, simultaneous or time-delayed addition, in the culture medium of said reconstituted embryo, at least one protein kinase inhibitor, preferably 6-DMAP, and at least one inhibitor of protein synthesis, preferably cycloheximide (CHX). Preferably, said activation is carried out by simultaneous addition of 6-DMAP and CHX for an activation duration which preferably extends from 30 minutes to 2 hours, preferably one hour.

It is also one of the objects of the present invention to provide a method of producing a recombinant protein by a transgenic animal, in particular rabbit, obtained by a method according to the invention. The transgene which codes for said recombinant protein is not limited to a particular DNA sequence. The DNA sequence of the transgene may be of purely synthetic origin (for example routinely carried out using a DNA synthesizer), or may be derived from mRNA sequences by reverse transcription, or may be derived directly from sequences of genomic DNA. When the DNA sequence is derived from RNA sequences by reverse transcription, this may or may not contain all or part of non-coding sequences such as introns, depending on whether the corresponding RNA molecule has undergone, partially or totally , splicing. The transgene can be as small as a few hundred base pairs of cDNA or as large as a hundred thousand base pairs of a gene locus comprising the exon-intron coding sequence and the regulatory sequences necessary for obtaining a spatio-temporally controlled expression. Preferably, the recombinant DNA segment has a size of between 2.5 kb and 1000 kb. Either way, the recombined DNA segments can be less than 2.5 kb and greater than 1000 kb. The transgene or DNA sequence of the present invention is preferably in native form, i.e. derived directly from an exogenous DNA sequence naturally present in an animal cell. This DNA sequence in native form can be modified for example by insertion of restriction sites necessary for cloning and / or by insertion of site-specific recombination sites (lox and flp sequences). Alternatively, the DNA sequence of the present invention may have been created artificially in vi tro by recombinant DNA techniques, for example by combining portions of genomic DNA and cDNA.

The transgene which codes for said recombinant protein preferably contains regulatory sequences suitable for directing and controlling the expression of the genes encoding said polypeptides in the appropriate cell type (s). The term “elements controlling gene expression” is intended to denote all the DNA sequences involved in the regulation of gene expression, that is to say essentially the regulatory sequences for transcription, splicing and translation. Among the DNA sequences which regulate transcription, mention should be made of the minimum promoter sequence, the upstream sequences (for example, the SP1 box, the IRE for “interferon responsive element”, etc.), the activator sequences ( "Enhancers"), possibly the inhibitor sequences ("silencers"), the insulator sequences ("insulators"), the splicing sequences. The elements controlling gene expression allow either constitutive, ubiquitous expression, inducible, specific for a cell type ("tissue-specific") or specific for a stage of development. These elements may or may not be heterologous to the organism, or may or may not be naturally present in the genome of the organism. It is obvious that, depending on the desired result, those skilled in the art will choose and adapt the elements for regulating gene expression. To direct the expression of the transgene in an animal biological fluid, such as milk, the transcription regulatory sequence used is selected from the promoter sequences of the genes active specifically in the cells secreting these biological fluids, such as the gland cells. breast for example to direct expression in milk. Among the preferred biological fluids, mention should be made of milk, blood, semen, urine. Preferably, the recombinant protein according to the invention is secreted by the cells of the mammary glands in milk. Thus, the preferred promoter or promoter sequences are those which are both effective and specific in breast tissue. By effective is meant that the promoters are strong in breast tissue and can support the synthesis of large amounts of protein secreted in milk. Among these promoters, mention should be made of the promoters of casein, lactoglobulin, lactalbumin, which include, without limitation, the promoters α-, β-, and γ- casein, the promoter of α-lactalbumin and promoters of β-lactoglobulin. The preferred promoters come from rodents, mice or rats, rabbits, pigs, goats, sheep. So more preferred, the promoter is that of a whey acidic protein gene, and the most preferred WAP promoter is a rabbit WAP promoter described in US Pat. No. 5,965,788, the pig WAP promoter, mouse WAP promoter.

The use of a transgenic animal, in particular a transgenic rabbit, obtained by a process according to the invention for the production of recombinant proteins of interest, preferably in the milk of the animal, is an object of the present invention. The recombinant protein of interest can be any protein, for example a therapeutic protein such as α-, β—, δ-globin, blood coagulation factors (factors VIII and IX), cell surface receptors, antibodies, enzymes, etc. and other proteins necessary for, for example, correcting inherited or acquired defects in a patient. The invention also relates to the use of a transgenic animal, preferably a transgenic rabbit, capable of being obtained by the method according to the invention, as a model for studying human pathologies. As an example of human pathologies, mention should be made of cystic fibrosis, atherosclerosis, cancers, metabolic diseases, ocular pathologies. Given the genetic polymorphisms present in the population, it may be advantageous to analyze or obtain a physiological, pathophysiological or behavioral response characteristic that the transgenic animals according to the invention, and in particular the transgenic rabbits according to the invention have different genetic backgrounds. Thus, the rabbits according to the invention can be selected from the New-Zealand, Fauve-de-Bourgogne, Argenté-de-Champagne, Californiennes, Géant-de-Bouscat breeds, all breeds whose zoological specificities are defined in an official standard. (The purebred rabbit, Ed. 2000 French Federation of Cuniculture (Ed.) And their crosses, in particular those which give rise to commercial strains such as the strain GD22 / 1077.

Other characteristics and advantages of the present invention will be better demonstrated on reading the following examples.

FIGURES:

FIGURE 1: Confocal images of a reconstituted embryo (NT) at stage 1 cell ± mmunolabeled using an anti-alpha tubulin antibody (green) and DNA with propidium iodide (red). (AT). Before the second round of electro-stimulation, the chromatin appears condensed in the chromosomes and attached to the spindle; the arrows in the insert

(3 times enlarged view of the spindle region) indicates individual chromosomes near the spindle poles. (B). Following the withdrawal of CHX and 6-DMAP, 72% of the reconstituted embryos (NT) (n = 25) already have a small nucleus and a micro- tubular interphasic formed (arrow). (VS) . 1 hour after the withdrawal of the drugs, all the reconstituted embryos (NT) are in interphase and 71% (n = 17) of them present a single and large nucleus resembling the pronuclei as observed in rabbit zygotes. Bar = 50 μm.

FIGURE 2: Development of reconstructed rabbit blastocysts with cumulus cells or derived from eggs activated in vitro or fertilized in vivo.

(A), In vitro increase in the number of cells between days D3 and D4 of embryos recovered either directly from donors (in vivo, n = 27), or cultured from the cell stage (approximately 20 hours post HCG ), either after natural mating (in vitro, n = 44) or by nuclear transfer (n = 31, NT); (B), Average diameter and average length of the embryonic discs on day D8; (C), example of a reconstructed blastocyst (obtained by nuclear transfer) delayed and recovered on day D8 after a transfer to the 4 cell stage in an asynchronous recipient female (- 16 hours); the embryonic disc (large arrow) is visible but the blastocyst is still surrounded by a thin protective layer of the embryo

(small arrow) which should normally have disappeared on day D7 (Denker, 1981). In vivo fertilized embryos are harvested either directly from donors (in vivo) or following transfer to stage 1 recipient females cell (control). In vitro fertilized embryos are collected at the 1 cell stage (in vitro).

FIGURE 3: Schematic representation of the protocol used for the in vivo development of embryos reconstituted from cumulus donor cells. Only embryos transferred to the 4-cell stage in asynchronous females (at 10 p.m.) develop over time.

FIGURE 4: Rabbits born by nuclear transfer. (A), rabbit clone No. 0107 with the corresponding controls: A1, expression of the self-fluorescent protein eGFP (arrow head) detected by confocal microscopy from hair follicles obtained by an ear biopsy at 1 month; A2, same but the detection is carried out by light transmission microscopy; A3, amplification of the eGFP transgene (PCR 2) and exon 10 of the CFTR gene used as DNA quality control (PCR 1); this confirms that rabbit N ° 0107 (lines 8 and 9) and its progeny N ° 107B (who died 1 day after birth; lines 10 and 11) derive from donor cumulus cells (lines 12 to 13); B1-B2, 3 other rabbits from 2 other different litters; the rabbits in Bl have now proven to be fertile. EXAMPLES

1) MATERIALS AND METHODS

1.1) Source of oocytes and cumulus cells

Metaphase II (Mil) oocytes are collected from “New Zealand” rabbits superovulated by injections of FSH hormones followed by an injection of HCG hormone, coupled to a vasectomized male 16 hours after an injection of chorio-gonadotropin Human (hCG). The oocytes are then incubated in 0.5% hyaluronidase for 15 min (Sigma) to remove the cumulus cells by gentle pipetting. For nuclear transfer, the oocytes are enucleated as described above (Adenot et al., 1997). At the same time, cumulus cells are taken either from “New-Zealand” rabbits or F1 rabbits obtained by crossing between “New-Zealand” rabbits crossed with “Fauves de Bourgogne” rabbits or female rabbits "New Zealand" transgenic fl comprising a DNA construct with a coding sequence for the increased green fluorescence protein (eGFP) placed under the control of an EF1 promoter ("elongation factor 1") or an HMG promoter. EGFP fluorescence and PCR amplification reactions are used as markers of cumulus donor cells. The cumulus cells are then stored at 38 ° C. in PBS without calcium or magnesium supplemented with 1% PVP 40,000 (polyvinyl pyrolidone (PVP)) before their use as nucleus donor cells. 1.2) Activation of oocytes and nuclear transfer

To reconstruct the embryos by nuclear transfer (NT), individual cumulus cells are inserted by micro-manipulation under the pellucid zone of the enucleated oocytes. The embryos obtained by nuclear transfer (NT embryos) and the Mil oocytes are activated in the following manner: 2 phases of electrical stimulation are applied at 1 hour delay with a Grass stimulator (3 DC current draws of 3.2 kV x cm - ¹ for 20 μs each in 0.3 M mannitol containing 0.1 mM Ca ²⁺ and Mg ⁺ ), the first phase inducing the fusion of the oocyte and the cumulus cell. The reconstituted embryos are kept for one hour in a culture medium at 38 ° C. After the second phase, the NT embryos are incubated in the presence of cycloheximide (5 μg / ml) and 6-DMAP (2 mM) in M199 medium for 1 hour; oocytes are incubated with one or both of these drugs simultaneously for 1 hour. After extensive washing to remove the drugs, the cells are again re-cultured in a microdrop of 50 μl of B2 medium supplemented with 2.5% fetal calf serum (FCS) under mineral oil (Sigma M8410) at 38 ° C under an atmosphere saturated with 5% CO ₂ . This activation protocol is applied to NT embryos, approximately 18 to 20 hours after donor mating.

1.3) Analysis of the preimplantation stages The microtubular organization and chromatin in NT embryos at stage 1 cell are observed as previously described (Adenot et al., 1997), except that the state of fixation lasts 20 min at 37 ° C and that the mounting medium is from Vectashield (Vector Laboratories). The cleavage rates of NT embryos and parthenotes are evaluated from 9 p.m. to 11 p.m. after electro stimulation. Development rates up to the blastocyst stage are estimated after an in vitro culture of 3 to 4 days. For the evaluation of the number of cells, the embryos are fixed as described above, then stained with Hœchst 33442 at 1 μg / ml then mounted on well slides in Vectashield and analyzed under epifluorescence. The controls are blastocysts which have either developed in vitro from a 1 cell embryo collected from a superovulated rabbit or which have developed in vivo from non-superovulated females crossed with a male and then sacrificed 3 or 4 days later.

1.4) In vivo development:

The recipient females are crossed with a vasectomized male either at the same time or 16 hours or 22 hours after the crossing of the oocyte donor females with a vasectomized male. In the case of synchronous females (i.e. crossed at the same time as the oocyte donor females) the reconstituted NT embryos are transplanted in stage 1 cell 1 to 3 hours after activation or are transplanted in stage 4 cells after overnight culture. In the case of recipient females Asynchronous (that is to say crossed 16 hours or 22 hours after the female donors of oocytes), the reconstituted NT embryos are transplanted either at the stage 1 cell or at the stage 4 cells after an overnight culture. The embryos are surgically transplanted through the infondibulum into each of the oviducts of recipient females. The implantation rate of parthenotes and NT embryos is evaluated after sacrifice of the recipient females on day D8. Pregnancy is determined by palpation 13 or 14 days after embryo transplantation and pregnant recipient females are delivered by cesarean section 31 days after mating.

1.5) PCR analysis

The presence of GFP transgenic markers is detected by PCR using a primer-sense (SEQ ID No. 1) and an antisense primer (SEQ ID No. 2) (GENSET, France). To control the quality of the DNA, the PCR is carried out on 300 to 400 ng of DNA prepared with the tissue extraction kit (QIAGEN, USA) with the primer-sense (SEQ ID No. 3) and l antisense primer (SEQ ID No. 4) which covers exon 10 of the rabbit CFTR gene (GENSET, France). The amplifications are carried out with Taq Polymerase (Q.BIOGEN, France) through 35 amplification cycles, as follows: 94 ° C for 20 s, 57 ° C for 30 s and 72 ° C for 1 min. The size of the amplified fragments is 240 base pairs for the CFTR gene and 350 base pairs for the eGFP transgene. The PCR fragments are separated on a 1.5% agarose gel in TAE (Tris Acetate EDTA) and then stained with ethydium bromide and are compared to a 100 base pair scale used as a size marker (BIOLABS, England). The negative controls consist of double-distilled water and DNA from the recipient female, while the positive control corresponds to DNA from cultured transgenic fibroblast.

2) ACTIVATION, NUCLEAR APPEARANCE AND IN VITRO DEVELOPMENT OF RECONSTITUTED RABBIT EMBRYOS OBTAINED BY NUCLEAR TRANSFER

The inventors have previously described (Adenot et al., 1997) that oocytes from ovine rabbits aged in the oviducts before their collection (aging in vivo), form pronuclei during a period of one hour after activation by a stimuli: this corresponds to a time 3 times faster than when the freshly ovulated oocytes are cultivated up to the same age (aging in vitro).

When oocytes are activated in the presence of the protein phosphorylation inhibitor (6-DMAP), these oocytes form pronuclei more quickly than would older oocytes in vivo (unpublished data from the inventors). Thus, the activation conditions can significantly alter the timing of nuclear formation of rabbit zygotes. Conversely, other species of mammals, the rabbit zygotes have the particularity of entering the S phase very soon after activation (Szôllôsi, 1966). This indicates that the duration of metaphase II (Mil) until the interphasic transition should be carefully considered when establishing an activation protocol for nuclear transfer in this region. species. In somatic mammalian cloning, 6-DMAP or the inhibitor of protein synthesis cycloheximide (CHX) are often used following the activation of embryos obtained by nuclear transfer (NT) by agents increasing the concentration of intracellular calcium. These drugs promote the inactivation of cdc2 / cyclin-B and the ERK / MAP kinases involved in the arrest of oocytes at the Mil stage. Cycloheximide, however, also inhibits DNA replication in activated oocytes (Moos et al., 1996; Soloy et al., 1997) and 6-DMAP may also affect the kinase activity known to be involved in the regulation of cell cycle (Meyer et al., 1997). The inventors therefore considered that an incubation of 1 hour with CHX and / or 6-DMAP after activation of the oocytes could be sufficient for the rabbit species. Previous, unsuccessful experiments in somatic cloning of rabbits used a 6-DMAP incubation of 2 (Yin et al., 2000; Dinnyés et al., 2001) or 4 (Mitalipov et al., 1999) hours. In the present invention, the inventors have found that electrically activated Mil oocytes exposed for 1 hour to CHX (n = 48), 6-DMAP (n = 48) or to a mixture of the 2 drugs (n = 130) efficiently cleave at the 2 cell stage (94%, 96% and 100% respectively). However, it develops at the blastocyst stage significantly better when they are exposed simultaneously to CHX and 6-DMAP (89%) or to 6-DMAP alone (79%) than to CHX alone (50%) (Table 1 ). These rates are higher (Dinnyés et al., 2001; Mitalipov et al., 1999) or similar (Yin et al., 2000) than those reported by other researchers who use 6-DMAP to treat oocytes for 2 hours after electrostimulation (Yin et al., 2000; Dinnyés et al., 2001) or for 4 hours after electrostimulation (Mitalipov and al., 1999; Dinnyés et al., 2001) or who use ionomycin (Mitalipov et al., 1999).

The inventors used an activation protocol using a CHX / 6-DMAP mixture to reconstruct NT embryos with a freshly collected nucleus of cumulus cells. The inventors chose this type of cells, considered to be stopped at the G1 / G0 stage of their cell cycle, because these cells were initially used as a model to demonstrate the feasibility of somatic cloning (Wakayama et al., 1998; Wells et al., 1999). Observations under the confocal microscope of NT embryos fixed just before the second phase of electro-stimulation show that the c romatin is condensed into chromosomes and associated with the spindle (N = 14; fig. 1A). This condensation results from the high MPF activity in the recipient cytoplast (Campbell et al., 1996). However, a typical arrangement of chromosomes characteristic of Mil oocytes was not observed. Instead, collapsed chromosomes form a misaligned metaphasic plateau, and individual chromosomes are sometimes seen near the spindle poles (Fig. 1A, insert). This unusual chromic structure, probably linked to the nuclear stage of the donor nucleus (Wakayama et al., 1998) is compatible with development to completion in mice (Wakayama et al., 1998; Wakayama et al., 1999) . After Having eliminated the drugs, the inventors observed that 72% of the NT rabbit embryos (N = 25) already exhibited an interphasic chromatin and a microtubular organization (FIG. 1B). 1 hour later, all the embryos were in interphase and 71% of them (N = 17) showed a large and unique pronuclear structure (fig. 1C) like that observed in rabbit zygotes (data not shown). From these observations, the inventors have concluded that the metaphase II to interphase transition is rapid in reconstituted NT embryos and lead to an apparently normal remodeling of the foreign chromatin by the cytoplasm of the oocyte.

When the NT embryos are left in culture (n = 135), 93% of them cleave at the two-cell stage and 47% of them develop into blastocysts. The pre-implantation development rates previously obtained in somatic cloning experiments in rabbits were much lower (16 to 30%) whether with cumulus cells (Yin et al., 2000) or donor fibroblasts (Dinnyes et al., 2001; Mitalipov et al., 1999).

NT embryos reach the blastocyst stage im vi tro on day D3 (D3) as zygotes and parthenotes do, but their growth, determined by the counted number of cells, is slower, which means that these NT embryos have development on day D4 of about 1 day back

(fig. 2A).

3) IN VIVO DEVELOPMENT OF RABBIT EMBRYOS OBTAINED BY NUCLEAR TRANSFER In rabbit species, the in vivo development of blastocysts is rapid and important. This development spreads to the surrounding walls of the uterus, so that the individual position of the blastocysts quickly becomes recognizable as implantation sites from the J6 stage on the uterine horns of sacrificial recipient females (Denker, 1981). The inventors have found that embryos obtained by nuclear transfer can form implantation sites for a transfer at stage 1 or 4 cell (s) into the uterus of synchronous recipient females (i.e. crossed with a male vasectomized at the same time as females donating nuclei). However, the number of implantation sites for blastocysts obtained by nuclear transfer (NT) is less important than that of controls and parthenotes (Table 2); no embryonic structure obtained by NT can be demonstrated following the dissection of the uterine horns on day D8.

When NT embryos at the 4-cell stage are transferred into cross-asynchronous recipient females 16 hours after the donor females (Fig. 3), the implantation rate increases (Table 2) and is not significantly different from that obtained with the controls (synchronous transfer) or with parthenotes (synchronous or asynchronous transfer). Under these conditions, the inventors were able to collect embryos at the advanced blastocyst stage; these embryos have a flat embryonic disc (fig. 2C large arrow) of about 1.1 mm in length (n = 8, range 0.8 - 1.5 mm) and all but one are still clearly surrounded by a thin protective layer of extracellular material (small arrow, fig. 2C) which was gradually affixed to the surface of rabbit embryos throughout the transit through the female genital tract. The dissolution of these muco-substances depends on the synergistic actions of the blastocysts and the endometrium (Denker et al., 1975) and contributes to generating a very narrow window of implantation in this species. When these embryos are examined carefully from the abembryonic pole, no apparent disorganization of the cover can be observed indicating that the blastocysts obtained by nuclear transfer were equivalent at least to normal embryos at the J7 stage (Denker, 1981). None of the recipient females transplanted, either synchronously or asynchronously (16 hours), can be diagnosed pregnant at mid-gestation (Table 3), even when they have been transferred to unmanipulated "helper" embryos of another breed. rabbit (Burgundy big cats; data not shown) or when an excess of embryos obtained by nuclear transfer has been transferred to them (up to 39 per female, data not shown). These observations suggest that only very few blastocysts obtained by nuclear transfer can implant because their development is not delayed. This was confirmed by the fact that, when carrying out an examination on day D8, the inventors can observe in one case a blastocyst obtained by nuclear transfer already adherent to the uterine epithelium (data not shown), this blastocyst being very similar in size at the control normally installed (range 1.1 to 2.6 mm, fig. 2B). Although they are smaller than normal embryos in vivo at the J8 stage (Gottschewski et al., 1973) (fig. 2B), these controls can normally develop until term following a transfer to stage 1 or 4 cell (s) in synchronous recipient females (data not shown).

The inventors have thus extended the asynchrony between donor females and recipient females from 4 pm to 10 pm (fig. 3). Such marked asynchrony at early stages of developmental cleavage has never been achieved in the past with embryos obtained by nuclear transfer and is compatible with a term development of zygotes. Under these conditions, 10/27 (37%) of the asynchronous recipient females (22 hours) were diagnosed pregnant after palpation on day D14. From these females, 4 (40%) gave birth on day D31 to 6 live rabbits (fig. 4) weighing 30 to 90 g (average weight: 65 g). Such variability is also observed when the rabbits are born from a litter of a reduced size (from 1 to 4 fetuses) which occurs occasionally in the pet store, especially when the rabbits come from micro-injected embryos with DNA solutions at the pro-nucleus stage. The expression of the transgenic marker eGFP from the hair follicles from birth (fig. 4) and from lymphocytes obtained at about 1 month of age (data not shown) confirms that the rabbits result from nuclear cell transfer - cumulus. Rabbits with a normal morphological appearance (respective weights: 90 and 30 g) died 1 day after birth and, for one among them, the inventors suspect a deficiency in the process of adoption by the nursing mother. The other 4 young rabbits developed normally and two of them (fig. 4, bottom left) reproduced and gave birth to 7 and 8 healthy young rabbits respectively following a natural crossing.

The present invention overcomes the limitations encountered when cloning certain mammal species considered difficult to clone so far, such as the rabbit (Solter 2000). These limitations can be overcome by taking into account the seemingly tenuous differences between the development of the oocyte and early embryo. The results presented by the inventors therefore indicate that somatic cloning can be carried out successfully in any species of mammal. Surprisingly, shortened timing, conventional activation procedures and an asynchromy of almost 1 day when transferring the reconstructed embryos into delayed recipient females compensate for the developmental delay which already exists at the time of the first cleavage and seems to have an effect. decisive in obtaining rabbit embryos obtained by nuclear transfer.

The process for obtaining rabbits by nuclear cloning is of great industrial interest, in particular for obtaining transgenic rabbits expressing a protein of interest, or for the genesis of animal models of human pathologies. Table 1. Effect of different treatments on in vitro development after parthenogenic activation of rabbit oocytes (CHX: cycloheximide; 6-DMAP: 6-dimethylaminopurine) FR03 / 00064

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Table 2. Implantation after transfer in synchronous and asynchronous recipient females (-16 hours)

Females Number of sites Number of Type of recipients of implantation blastocysts

Type pregnant females to / Number of implanted embryos / recipient embryos day D8 / Total transferred in number of female females

Receiving female recipients Receiving pregnant pregnant (%) transferred

Synchronous Control 5/6 15/54 (27.8%) 9/9

Parthenotes Synchronous 8/20 17/78 (21.8%) 0/1

NT Synchronous 5/16 7/91 (7.7%) 0

Asynchronous parthenotes 5/9 15/44 (34.1%) 3/3

Asynchronous NT 6/13 12/59 (20.31 1/7

Table 3. In Vivo Development of Reconstructed Rabbit Embryos by Somatic Nuclear Transfer

Type of recipient females Synchronous Asynchronous Asynchronous (-16h) (-22h)

1-cell 1-cell 4-cell

Stage of embryos

Number of reconstituted embryos

554 523 775 [Number of replicates] [19] [18] [27]

Number of embryos fused

427,346,612 [% from reconstituted embryos] [77.1] [66.2] [79.0]

Total number transferred

367 346 371 [% from fused embryos] [100.0] [100.0] [60.6]

Number of recipient females

19 18 27 transferred

Number of pregnant recipient females on day D14 [of 10 females transferred] [37.0]

Number of recipient females with 4 * calving [% of females transferred] [14.8]

Number of rabbits born 6 [% of embryos transferred] [1.6]

Number of young rabbits at weaning

Average weight of young rabbits at birth (in gr) 65 ± 20 ⁺

* abortions between days D15 and D29 of pregnancy (13 cotyledons and degenerated fetuses were recovered)

+ average weight of rabbits at birth in the inventors' pet store, for litters of equivalent number: 55.8gr ± 17.0 REFERENCES

Adenot et al. (1997) Mol. Reprod. Dev. 46, 325-336.

Baguisi et al. (1999) Nature Biotechnol. 17, 456-461. Bromhall et al. (1975) Nature 258, 719-722.

Campbell et al. (1996) Rev. Reprod. 1, 40-46.

Chen et al. (2001) Mol. Biol. Evol. 18, 1771-1788.

Denker et al. (1975) Cytobiol. 11, 101-109.

Denker (1981) Adv. Anat. Embryol. Cell Biol. 53, 123p.

Dinnyés et al. (2001) Biol. Reprod. 64, 257-263.

Evans et al. (1981) Nature 292, 154-156

Fan et al. (1999) Pathol. Int. 49, 583-594.

Gottsche ski et al. (1973) Schaper, Hannover. Graur et al. (1996) Nature 379, 333-335.

Hoeg et al. (1996) Froc. Natl. Acad. Sci. USA 93, 11448-11453.

Kennely and Foote (1965) J. Reprod. Fert. 9, 177-188. McGrath and Solter (1984) Science 226, 1317-1319.

Meyer et al. (1997) Methods Enzymol. 283, 113-128.

Mitalipov et al. (1999) Biol. Reprod. 60, 821-827.

Moos et al. (1996) J. Cell Sci. 109, 739-748.

Ozil and Huneau (2001) Development, 128, 917-928. Polejaeva et al. (2000) Nature 407, 86-90.

Soloy et al. (1997) Biol. Reprod. 57, 27-35.

Solter (2000) Nat. Rev., Broom. 1: 199-207.

Stinnackre et al. (1997) LM Houdebine ed., Harwood Académie Publishers, pp. 461-463. Szôllδsi (1966) Anat. Rec. 154, 209-212. Wakayama et al. (1998) Nature 394, 369-374.

Wakayama et al. (1999) Proc. Natl. Acad. Sci. USA 96, 14984-14989.

Wells et al. (1999) Biol. Reprod. 60, 996-1005. Whitfield et al. (1985) Control of Animal Cell Proliferation 1, 331-365.

Wilmut et al. (1997) Nature 385, 810-813.

Yin et al. (2000) Theriogenology 54, 1460-1476.

Claims

1. Method for producing embryos of non-human mammals comprising the following steps:

(a) Evaluate and / or determine the development asynchrony (T) between two embryos of the same species and of the same age:

(i) the first embryo being produced by crossing at time t ₀ of a male, preferably vasectomized, with a female having preferably received a hormonal treatment to increase ovulation, said first embryo being at least cultivated and / or manipulated in vitro; (ϋ) the second embryo being produced by crossing at time to of a fertile male with a female having preferably received hormonal treatment to increase ovulation, the said second embryo being normally fertilized or obtained by parthenogenetic activation, the evaluation and / or the determination taking place at the latest on the day of uterine implantation of said second embryo and;

(c) optionally, allow the said embryo transferred to step b) to implant and develop in the uterus of the said recipient female. T FR03 / 00064

47

2. Method according to claim 1, characterized in that said first embryo is cultured and / or handled in vitro at the latest until the day of implantation.

3. Method according to claims 1 to 2, characterized in that the evaluation and / or determination is carried out at a stage of development chosen from, stage 1 cell, stage 2 cells, stage 4 cells, stage 8 cells, 16 cell stage, morula stage, blastocyst stage.

4. Method according to claim 3, characterized in that the evaluation and / or determination is carried out at the blastocyst stage.

5. Method according to claim 1 to 4, characterized in that the evaluation and / or determination of the development asynchrony T is carried out by cell counting.

6. Method according to claims 1 to 5, characterized in that said development asynchrony T is at least 15 hours.

7. Method according to claim 6, characterized in that said development asynchrony T is 24 hours.

8. Method according to claims 1 to 7, characterized in that the said embryo transferred to stage b) is cultivated under the same conditions as said first embryo.

9. Method according to claims 1 to 8, characterized in that said embryo transferred to step b) is at the 1 cell stage.

10. Method according to claims 1 to 8, characterized in that said embryo transferred to step b) is in the 2 cell stage.

11. Method according to claims 1 to 8, characterized in that the said embryo, transferred to step (b), is at the 4 cell stage.

12. Method according to claims 1 to 11 characterized in that said transferred embryo develops into a fetus.

13. The method of claim 12 characterized in that said fetus develops into a newborn.

14. Method according to claims 1 to 13, characterized in that said embryo cultivated and / or manipulated in vitro is a transgenic embryo.

15. Method according to claims 1 to 13, characterized in that said embryo cultivated and / or manipulated in vitro is a reconstituted embryo obtained by nuclear transfer. T FR03 / 00064

49

16. Method according to claims 1 to 13, characterized in that said embryo cultivated and / or manipulated in vitro is a reconstituted transgenic embryo obtained by nuclear transfer.

17. Method according to claims 1 to 16, characterized in that the said mammal is selected from rodents, lagomorphs, ungulates, equines, primates, except man.

18. The method of claim 17, characterized in that said mammal is a rodent selected from mice, rats, hamsters, guinea pigs.

19. The method of claim 17, characterized in that said ungulates is selected from cattle, sheep, goats, pigs.

20. The method of claim 17, characterized in that said lagomorph is rabbit.

21. Mammalian embryo except man and / or fetus, newborn baby, adult mammal, or cells derived therefrom, produced by a process comprising or including the process according to one of claims 1 to 20.

22. Embryo of transgenic mammals except humans and / or fetuses, newborns, adult mammals or cells derived therefrom, produced by a process comprising or including the process according to one of claims 1 to 20.

23. Mammalian embryo, except man, reconstituted in vitro obtained by nuclear transfer, and / or fetus, newborn, adult mammal, or cells derived therefrom, produced by a process comprising or including the process according to 'One of claims 1 to 20.

24. Descendants of said adult mammal according to claims 21 to 23.

25. An in vitro method for cloning mammals by nuclear transfer comprising or including a method according to any one of claims 1 to 20.

26. A method of producing rabbit embryos comprising the following steps: a) evaluating and / or determining the development asynchrony (T) between two rabbit embryos of the same age: the first embryo being produced by crossing at time t ₀ of a male preferably vasectomized with a female having preferably received a hormonal treatment to increase ovulation, the said first embryo being at least cultivated and / or manipulated in vi tro; the second embryo being produced by crossing at time t ₀ of a fertile male with a female having preferably received hormonal treatment to increase ovulation, the second embryo normally being fertilized or obtained by parthenogenetic activation; the evaluation and / or determination taking place at the latest on the day of uterine implantation of said second embryo normally fertilized or obtained by parthenogenetic activation; and b) transfer a rabbit embryo cultivated and / or manipulated in vitro, not older than the blastocyst stage into the uterus of a recipient female having been crossed with a vasectomized male at time t = t ₀ + T (+ / - 25% T); c) optionally, leave the said embryo transferred in step b) to implant and develop in the uterus of the said recipient female.

27. The method of claim 26, characterized in that the evaluation and / or determination is carried out at a stage of development between the days Dl and D7 post coitum.

28. Method according to claim 27, characterized in that the evaluation and / or determination is carried out on days D5 post coitum.

29. Method according to claims 26 to 28, characterized in that said development asynchrony T is 23 hours +/- 25%.

30. Method according to claims 26 to 29, characterized in that said embryo cultivated and / or manipulated in vitro is a transgenic embryo.

31. Method according to claims 26 to 29, characterized in that the said embryo cultivated and / or manipulated in vitro is a reconstituted embryo obtained by nuclear transfer.

32. Method according to claims 26 to 29, characterized in that said embryo cultivated and / or manipulated in vitro is a reconstituted transgenic embryo obtained by nuclear transfer.

33. Method according to claims 26 to 32, characterized in that said embryo transferred to step b) is at the 1 cell stage.

34. Rabbit and / or fetus, newborn, adult rabbit, or cells derived therefrom, produced by a process comprising or including the process according to one of claims 26 to 33.

35. Embryo of transgenic rabbit and / or fetus, newborn baby, adult rabbit or cells derived therefrom, produced by a process comprising or including the process according to one of claims 26 to 33.

36. Rabbit embryo reconstituted in vitro obtained by nuclear transfer and / or fetus, newborn, adult rabbit, or cells derived therefrom, produced by a process comprising or including the process according to one of claims 26 to 33 .

37. Descendants of said adult rabbit according to claims 34 to 36.

38. An in vi tro method of cloning a rabbit by nuclear transfer comprising or including a method according to any one of claims 26 to 33.

39. In vi tro method of rabbit cloning by nuclear transfer comprising the steps of: a) insertion of a rabbit donor cell or of a rabbit donor cell nucleus into an enucleated rabbit oocyte under conditions allowing obtain a reconstituted embryo; b) activation of the reconstituted embryo obtained in step a); c) transfer of said reconstituted embryo to a carrier rabbit, so that the reconstituted embryo develops into a fetus, and possibly into a newborn; and characterized in that the method comprises or includes a method according to any of claims 26 to 33.

40. The method of claim 39 characterized in that the transfer of nucleus into the recipient cytoplasm is carried out by fusion of the donor cell and the recipient cytoplasm.

41. Method according to claim 39 characterized in that the transfer of nucleus into the recipient cytoplasm is carried out by micro-injection of the donor nucleus into the recipient cytoplasm.

42. Method according to claim 39 characterized in that the said activation phase during the in vitro culture is carried out by simultaneous, successive or time-shifted addition, to the culture medium of the said reconstituted embryo, of at least a protein kinase inhibitor and at least one protein synthesis inhibitor.

43. In vitro method for cloning a mammal, non-human, comprising the steps of: a) insertion of a donor cell or of a nucleus of a donor cell into an enucleated oocyte of mammal of the same or a different species that of the donor cell, under conditions making it possible to obtain a reconstituted embryo; b) activation of the reconstituted embryo obtained in step a); c) transfer of said reconstituted embryo into a female mammal carrier, so that the reconstituted embryo develops into a fetus, characterized in that said activation is carried out by simultaneous, successive or time-delayed addition to the culture medium said reconstituted embryo, at least one protein kinase inhibitor and at least one protein synthesis inhibitor.

44. The method of claim 43 characterized in that said mammal is selected from rabbits, rodents, in particular rats, mice, and among cattle, sheep, goats, pigs, equines, primates, except humans.

45. Process according to claim 42 to 44, characterized in that the said protein kinase inhibitor is 6-DMAP and the said protein synthesis inhibitor is cycloheximide (CHX).

46. A method of producing a recombinant protein by a transgenic animal comprising the step of producing a non-human mammalian embryo according to claims 1 to 20.

47. A method of producing a recombinant protein by a transgenic rabbit comprising the step of producing a rabbit embryo according to claims 26 to 33.

48. Use of a transgenic animal capable of being obtained by the process according to claims 1 to 20 or of a transgenic rabbit capable of being obtained by the process according to claims 26 to 33 as a model for studying human pathologies .

49. Use of a transgenic animal capable of being obtained by the process according to claims 1 to 20 or of a transgenic rabbit capable of being obtained by the process according to claims 26 to 33 for the production of recombinant proteins.

50. Use according to claim 49 characterized in that said recombinant protein is produced in the milk of the transgenic animal.