EP1108052A1 - Method for remodelling an animal genome by zygotic transfer of a site-specific recombinase - Google Patents

Abstract

The invention concerns a method for transferring into a mammal's oocyte a site-specific, in particular Cre-mediated, recombinase in its active form. Said recombinase is expressed under the control of a specific promoter in male germinal cells comprising at least a specific site and remains associated with the spermatozoid chromatin. Said method is particularly useful for remodelling an animal genome, for instance for inserting a foreign sequence into an animal genome, and for replacing a sequence by another. In the event where said promoter is a Sycp 1 promoter, an application mode enables to recombine paternal and maternal chromosomes. The invention also concerns animals obtainable by said method, and the use of said transgenic animals in pharmaceutical, cosmetic or agri-food industries.

Description

 METHOD FOR RESHAPING THE GENOME OF AN ANIMAL BY ZYGOTIC TRANSFER OF A SITE SPECIFIC RECOMBINASE.

The present invention relates to a method for transferring a site-specific recombinase, in particular Cre, into its active form into an oocyte of a mammal. Said recombinase is expressed under the control of a specific promoter in male germ cells having at least one specific site and remains associated with sperm chromatin. This method is particularly useful for reshaping the genome of an animal, for example for inserting a foreign sequence into the genome of an animal, and for replacing one sequence with another. In the case where said promoter is the Sycpl promoter, a mode of application makes it possible to carry out recombinations between paternal and maternal chromosomes. The present invention also relates to animals capable of being obtained by this process, and to the use of these transgenic animals in the pharmaceutical, cosmetic, and food-processing industries.

The site specific recombination process represents a powerful tool for making predetermined changes in the animal genome. Several recombination enzymes, including the recombinase Cre of the bacteriophage PI (Sauer & Henderson, 1989, 1990 and Porter, 1998) catalyze a recombination event between two defined double-stranded deoxyribonucleotide sequences (LoxP target sequence for the Cre enzyme). These Cre recombination operations were carried out in cultured cells (Sauer and Henderson, 1990) and in plant cells (Albert et al. 1995), but not on the germ line of an animal.

The recombination reaction was most often used for the targeted "knock-out" deletion of specific genomic regions known as "floxed" by intramolecular recombination between two LoxP sites of the same molecule. In particular, deletions in specific cell types have been carried out from various genetic constructions making it possible to express specifically the Cre recombinase (Brocard et al. 1997). Targeted "knock-in" integration could represent a possible application of site-specific recombination and would be of great interest, but certain limitations did not allow such development. In general, a recombinase could catalyze intermolecular recombination between two DNA molecules each carrying a specific site (or recombination site) and would serve for the integration of a heterologous DNA (introduced into the cell) into a defined site ( a region of the cell's genome).

Application to the germ line of a mammal could be envisaged in mammals by the methods currently available, but the operation would require a series of long technically difficult steps, involving the selection of recombinant clones in ES cells in culture, l 'germinal chimeras, and a series of crosses before obtaining a stable recombinant line. On the one hand, the time required for all of these operations would be calculated in months, if not in years, and the yield would be low. On the other hand, the selection of recombinant clones would require the introduction of antibiotic resistance genes. These selections could introduce unpredictable distortions of gene expression.

In the current state of the art, mutations introduced by homologous recombination or excision are frequently lethal to animals. Therefore, the effect of these mutations in the male germ line cannot be assessed since the cyclic process of spermatogenesis involves a number of regulatory genes which perform critical functions in other organs. An example of this situation has been described for the Brcal gene (Gowen et al. 1996, Zabludoff et al. 1996). This limitation can be overcome by establishing spatial and temporal control of the expression of the recombinase. The use of specific tissue promoters and inducible promoters, the use of adenoviral vectors and forms of recombinase activated by hormones have been considered to alleviate this problem (Akagi et al. 1997, Brocard et al. 1997, Gu et al. 1994, Kuhn et al. 1995, Porter, 1998, Wagner et al. 1997).

These problems are solved by the present invention since recombinase is only expressed in male germ cells. Unexpectedly, it remains associated with sperm chromatin and is transferred by sperm into oocytes in active form after fertilization. This ultimately allows the genome to be reshaped.

An additional application could be site-specific homologous meiotic recombination between paternal and maternal chromosomes. This would require the presence of the enzyme during the first division of meiosis (essentially at the pachytene stage), which is precisely the case in the experiments leading to the present invention.

Meiosis reduces the size of the genome and generates genetic diversity by homologous recombination, while maintaining genetic integrity through a repair process. Little is known about the transcriptional regulatory processes in the early stages of meiosis. However, studies with transgenic mice have recently led to the identification of promoters specific for male cells, for example HSP70-2 (Dix et al. 1996), Pdha-2 (Iannello et al. 1997), Pgk2 (Robinson and al. 1989), Tcp-lObt (Ewulonu et al. 1993) and Hox-a.4 (Berhinger, 1993).

Addressing Cre expression to the germ line, published in

O'Gorman et al. 1997, uses the protamine promoter Prml, which is active only after meiosis. During the experiments described in this publication, no zygotic transfer of Cre was observed. However, in the context of the present invention, it has been found that the transfer can be demonstrated as soon as a specific LoxP site is present in the genome of the spermatozoa.

The Sycpl gene has been identified, cloned and located on chromosome 3 of the mouse (Sage et al. 1997). Protein is a major component of the central element of the synaptonemal complex (Meuwissen et al. 1992). It is present exclusively during the meiosis of the male and the female from the beginning of the zygotene stage until the diplotene stage (Meuwissen et al. 1992, Heyting et al. 1989, Offenberg et al. 1991, Dietrich et al. 1992, Moens et al. 1992, Dobson et al. 1994). Concomitant expression of mRNA and protein shows that the regulation of expression is at the transcriptional level (Meuwissen et al. 1992).

Unlike the above-mentioned promoters, which are expressed at relatively late stages of germline differentiation, Sycpl has the advantage of being expressed during the early phase of sperm meiosis. It was necessary to identify and select the elements responsible for the specific expression of Sycpl. Since it was not possible to establish a cell line which expresses the endogenous gene, experiments using transgenic mice were carried out. Thus, it has been found that a region of the mouse Sycpl promoter, located up to 260 bp upstream from the site of initiation of transcription of the Sycpl gene, is not only sufficient to direct the expression of a transgene during the initial period of meiosis in males, but is also the most effective in males without being active in the ovary. This Sycpl region is therefore useful for meiotic recombination between LoxP sites either carried on the same chromosome or at separate locations of the paternal and maternal chromosomes.

All the operations necessary for the implementation of the present invention require only a series of microinjections and reimplantation, a technique which is now routine, on a limited number of fertilized eggs (a range is most often sufficient). ). Unlike an operation carried out using ES cells, the procedure presented does not involve the use of selection systems, and the time required to obtain the recombinant mouse is a few weeks, which is considerably faster than for the selection of recombinants. In addition, the transgenic animals obtained and their progenies are completely stable.

Thus, none of the documents of the prior art describes or suggests the present invention as defined below. Description

Thus, the present invention relates to a method of inserting a foreign sequence into the genome of an animal comprising the following steps: a) expression of a site-specific recombinase in the spermatocytes of a male carrying at least one site specific in its genome, said recombinase remaining associated with sperm chromatin. b) Fertilization of the oocytes and transfer of the recombinase by said spermatozoa. c) Microinjection of a recombinant DNA having the structure “specific site- foreign sequence” into the male pronucleus after fertilization. d) Insertion of the transgene of interest at the specific site in the genome in the male pronucleus of the embryo at stage 1 cell, catalyzed by the recombinase released during the decondensation of the chromatin of the spermatozoon.

Advantageously, said recombinase is Cre and the specific site is a LoxP site.

In this process, said foreign sequence comprises a gene of interest, to be expressed under the control of a promoter, optionally, tissue (s) specific. The gene of interest can also have a splice acceptor element (SA), an IRES (Internai Ribosome Entry Site) sequence and / or a polyadenylation signal (for example AATAAA) and the gene of interest can code for a protein of interest or be a reporter gene for identifying specific tissue promoters.

In another aspect, the subject of the invention is a method of replacing a sequence X by a sequence Y in the genome of an animal which consists in crossing a male of genotype “specific site-sequence X- specific site” whose the spermatocytes express a site-specific recombinase, said recombinase remaining associated with sperm chromatin, and then by microinjecting a molecule of structure “specific site -sequence Y- specific site” into its genome, said recombinase catalyzing the excision of the sequence X during the meiosis of the spermatocytes and the integration of the sequence Y in the specific site just after fertilization of the oocytes. By sequence X or sequence Y is meant any coding or non-coding sequence. This method can be used for the inactivation of a gene of sequence X. According to the methods mentioned above and for the remainder of the description of the invention, said recombinase can be Cre and said specific site a LoxP site and be expressed under the control of a promoter allowing efficient expression in germ cells of a mammal. During transfer, Cre therefore remains associated with chromatin.

Any Cre protein, its mutants, and variants, and any mutant and variant of the LoxP sequence, can be used in the context of the present invention. Cre, well known to a person skilled in the art, has been described in particular in EP 300 422 (page 4 lines 5-33) and EP 220 009 (page 3 lines 31-39), incorporated in the description by reference. Numerous plasmids containing the Cre sequence are disclosed there and have been deposited in the American Tissue Culture Collection such as BSY90 (accession number ATCC 53255) and pBS39 (accession number ATCC 20772). The Cre sequence can be isolated from pBS39 by the restriction enzymes Xhol and Sali. Other Recombinase / site specific systems can be used in the context of the present invention. Mention may in particular be made of the FLP / FRT system derived from Saccharomyces cerevisiae, the S / RS system of Zygosaccharomyces rouxii and the Gin / gix system derived from bacteriophage Mu which are described in EP 814 165, incorporated in the description by reference.

The present invention also relates to a method for transferring into a oocyte a site-specific recombinase, in particular Cre, in its active form. This process consists in expressing in recombinant male germ cells the recombinase having a strong affinity for its site of action on DNA, said protein remaining associated with sperm chromatin at its specific site (for example at its LoxP site in the case of Cre), to fertilize oocytes and to transfer the protein by said spermatozoa. The protein is then released in its active form during the decondensation of the chromatin after fertilization.

Among the promoters which allow efficient expression in the germ cells of a mammal, there are in particular the promoters Sycpl (accession number in the databases U 35665), Tcp-lObt (M 84175), Pgk-2, HSP- 70-2, Pdha-2 or Prml (X 07626), or at least one region of these promoters. For example, said promoter may comprise at least one sequence having at least 80% identity with the -260 / + 5 region (SEQ ID No. 1) of the mouse Sycpl promoter. This region is sufficient to effectively induce the expression of a gene of interest specifically during early meiosis of the spermatocytes of a mammal.

Examples of promoters which can serve for the accomplishment of the present invention are illustrated in Table 1 below. The references of the table are incorporated by reference into the description.

Table 1: Promoters active at different stages of spermatogenesis.

The present invention also relates to transgenic animals capable of being obtained by the methods described above.

Another object of the present invention is a nucleotide sequence making it possible to effectively promote the expression of a gene of interest during the first meiotic phase of the spermatocytes of a mammal, characterized in that it comprises at least one sequence having at least 80% identity with the sequence SEQ ID No. 1. Preferably, said nucleotide sequence comprises at least the sequence SEQ ID No. 1 or SEQ ID No. 2.

The -54 / + 5 region of the Sycpl promoter (SEQ ID No. 3) is the minimum sequence for addressing expression in spermatocytes. The -260 / + 5 region is however required for expression at a maximum rate.

Thus, the minimal nucleotide sequence of the Sycpl promoter is defined to promote efficient and addressed expression in spermatocytes as comprising at least one sequence having at least 80% identity with the sequence SEQ TD No. 1. SEQ ID No. 1 and SEQ ID No. 2 are the sequences -260 / + 5 in mice and -316 / + 5 in rats respectively. Preferably, this nucleotide sequence is characterized in that it comprises at least the sequence SEQ ID No. 1 or SEQ ID No. 2. It goes without saying that any sequence equivalent to the sequences SEQ ID No. 1 and SEQ ID No. 2 are covered by the present invention. The term “equivalent sequence” is intended to mean any sequence of a mammalian Sycpl promoter sufficient to effectively induce the expression of a gene of interest specifically in the spermatocytes of mammals, such as for example pigs, sheep, cattle, and rodents.

In a further aspect, the invention relates to a nucleotide sequence comprising at least one of said sequence fused to a sequence coding for a gene of interest. The gene of interest is preferably a gene coding for a site-specific recombinase, in particular Cre. It also relates to a DNA molecule comprising at least this sequence in the form of a plasmid, a viral vector, a pseudo-vector, or a linear or circular naked DNA.

The present invention also relates to a transgenic animal having in its genome said nucleotide sequence allowing the expression of a gene of interest specifically in spermatocytes, preferably a site-specific recombinase, in particular Cre. The recombinase remains associated with the chromatin of the spermatozoa. This animal is useful for the previously mentioned processes.

The present invention also relates to a method of recombination between paternal and maternal chromosomes which consists in crossing a male animal carrying in its genome one or more specific site (s), with a female animal carrying in its genome one or more site ( s) specific (s) characterized in that the recombinase, expressed during the first phase of meiosis, catalyzes the recombination between specific sites.

In another aspect, the invention relates to an animal capable of being obtained by this method and a multi-transgenic animal obtained by crossing the animals capable of being obtained by the methods described above. The transgenic animal according to the invention is a mammal, in particular selected from sheep, pigs, cattle, and rodents.

An advantageous aspect of the present invention relates to the use of said transgenic animal for the preparation of substances useful in medicine, in cosmetics, and / or in food industry, in particular of active substances, preferably peptides or polypeptides, antibodies, antigens, hormones, or growth factors, and / or for the manufacture of food supplements. For example, a protein of interest can be produced in mammalian milk under the control of a specific tissue promoter; for example WAP rabbit or WAP mouse (European patent application No. 92401635.5-2105).

Said animal can also be used as an experimental model for the identification of genes, promoters, proteins, in particular genes involved in genetic diseases, and / or for testing the action of active substances, for the preparation of organs or cells with better immunocompatibility with humans than wild-type organs or cells or capable of having a low rate of rejection by human immune defenses, and for breeding.

The present invention also relates to all the elements as described in the claims, said elements being incorporated in the description by reference.

For the rest of the description, reference is made to the legends of the figures presented below.

LEGENDS OF FIGURES

Figure 1: specific site recombination.

A- Schematic representation of the deletion of specific genomic region by intramolecular recombination between so-called floxed regions located between two LoxP sites. B- Targeted integration of a foreign sequence catalyzed by intermolecular recombination between two DNAs each carrying a LoxP site. Figure 2: homologous meiotic recombination between paternal and maternal chromosomes at a specific LoxP site.

Figure 3: activation of the LoxP-βgeo transgene without promoter after microinjection of the Pgkl-LoxP construct.

A. The plasmid pGK-LoxP comprises a BamHI-HindIII fragment, of PGKβgeobpA (Friedrich and Soriano, 1991) containing the promoter of the Pgkl gene, inserted between the BamHI (B) and HindIII (H) sites of pBS112 (Sauer and Henderson, 1990). The plasmid DNA was cleaved with HindIII and Pvul (P) before microinjection, which has the effect of deleting the LoxP site at 5 '. Microinjection was carried out according to standard techniques (Hogan, 1986) in fertilized eggs from the cross between a wild type female and a double transgenic male carrying the transgenes Tcp-lObt-cre and LoxP-βgeo.

B, C and D. The microinjected eggs were kept in the culture medium for 24 hours and were stained with X-Gal to detect the β-galactosidase activity. B is the control (no microinjection). No endogenous β-galactosidase activity is detected. C and D are two series of eggs into which the construction described in A has been microinjected. The β-galactosidase activity is detected (coloration and arrows).

Figure 4: GFP specific site insertion at the Rxrα locus.

A. Schematic representation of the genome of the parent male mouse Sycpl -Cre Rxr ^{ΛAF (LNL)} in somatic cells and in gametes.

B. Map of the LoxP-GFP insertion vector. The plasmid was constructed by successive insertions inside the plasmid pBS112 (pBR322 containing a LoxP site, Sauer and Henderson, 1990) of the following fragments:

- BamHI-NcoI from pGT1.8 IRES βgeo (Mountford, 1994) with a part of the En-2 intron of the "engrailed" gene of Drosophila, a splice acceptor (Sa), and an Ires (Internai Ribozomal entry site).

- HindIII-EcoRI from pEGFP (Clontech) containing the coding region for GFP.

Sacl-Xhol from pSAβgeo with the polyadenylation signal (pA). C. Diagrams representing the recombination carried out by Cre leading to integration into Rxrα ^{ΔAF2 ()} and the modified locus resulting from site-specific recombination.

D. Results of the PCR Amplification of the Genomic DNA of 13 Babies Obtained from the Microinjected Eggs Using the Primers GFP1 and GFP2, and the Primers RXR1 and Int2 (Shows the RXR-Int Junction. Presence or Absence Sycpl -Cre is verified by southern blot.

Figure 5: structure and sequence of the 5 'region of the mouse Sycpl gene

A- Schematic map of the 5 'region of Sycpl Exons 1 and 2 are numbered, the presence of the first ATG and the transcription initiation site (+1) are indicated. The limits of the fragments used for the construction of the different transgenic lines are shown. The restriction sites are: B for BamHI, P for Pstl, S for SacII, and A for Apal.

B- Comparison of the Sycpl sequence of the mouse (M) and the rat (R). Putative binding sites for transcription factors are indicated (boxed in rectangles). The transcribed sequence (exon 1) is underlined.

Figure 6: expression of the various Sycpl / luc constructions in a somatic cell line Each bar represents the average of three experiments, each carried out three times. Error bars indicate the standard error to the mean. The statistical values were calculated using the Student test for this figure and the others. All results relate to CMV / lacZ internal control. The control vector (pXPl) contains the reporter gene for luciferase without a promoter. The activity of the plasmid SV40-luc was arbitrarily fixed at 100.

Figure 7: the specific expression in the testes of reporter genes in transgenic lines [-722 / + 102] lacZ, [-260 / + 102] lacZ, [-260 / + 102] Iuc and [- 54 / + 102 ] luc. Each bar represents the average activity of at least five transgenic males from different lots. Error bars indicate the standard error to the mean. This activity is normalized to the quantity of proteins and to the background noise of the mice not transgenic belonging to the same batch. The low level of lacZ activity is due to a high endogenous level of β galactosidase type in testicular cells. For each of the chimeras, two lines were studied in detail (1 and 2). Te represents the testicles, Li the liver, Sp the spleen, Ki the kidneys, He the heart and Lu the lungs.

Figure 8: Expression of the reporter gene Iuciferase in the testes of transgenic mice [-54 / + 102] read and [-260 / + 102] luc during their development.

Liver extracts were used as a negative control. Each bar represents the average of at least four transgenic males from different lots. Error bars indicate the standard error to the mean. P8 to P14 on the x-axis represent the stages of development from the eighth day to the fourteenth day.

Figure 9: Expression of the reporter gene read in transgenic mice [-260 / + 5] in different organs.

The specific expression in the testes of the reporter gene Iuciferase in transgenic mice [-260 / + 5] luc is shown. Three independent lines were tested for the expression of the reporter gene. Each bar represents the average activity for at least five transgenic males. Error bars indicate the standard error to the average among experiments.

The legend of the various organs tested is identical to the legend in Figure 7.

Figure 10: Expression of the reporter gene Iuciferase in the testes and liver of mice belonging to the transgenic line [-260 / + 5] during development. The horizontally striped bars represent expression in the testes and the diagonal striped bars represent expression in the liver.

P8 to PI 5 represent the stages of development from the eighth day to the fifteenth day respectively. Error bars indicate the standard error to the average among experiments.

Figure 11: The Sycpl / lacZ transgenes are not expressed during meiosis in the female. PCR tests were carried out on the RNA coming from the testes of the liver of the fetal ovary El 7 of transgenic mice [-260 / + 102] lacZ and on the RNA of the fetal ovary El 6 of transgenic mice [- 1848 / + 102] lacZ. The RNA was pretreated with Dnasel and was subjected or not to digestion with RnaseA (+ and -) before the reverse transcription. Sycpl transcripts are detected in RNA from adult testes and RNA from the ovaries at the embryonic stage of day sixteen (EI 6) and the seventeenth day (EI 7), but in RNA of the liver. LacZ mRNA is only detected in transgenic adult testes.

Figure 12: Excision of floxed sequences in the offspring of a Sycpl- male

Cre / R rα ^ ™.

The Cre sequences identified by PCR amplification with a pair of primers Crel and Cre2 (Rxr wild allele), Rxra ^{ΔAF2 (L)} (deleted allele), and Rxr ^{ΔAF2 (LNL)} (target allele), with RXR1 and RXRII and tk- neo, with tkneol and tkneo2 (Feil et al. 1996). Column 1 corresponds to the progenitor mouse, and columns 2 to 10 represent babies belonging to the same litter.

Figures 13 and 14: targeted deletion of multicopy floxed genes in germ cells. The DNA was prepared from various tissues originating from male double transgenic progenitor and extracted from the tail of their descendants, digested with the restriction enzyme HindIII and using the plasmid DNA of pSAβgeo (column 1 to 7) or by pPGKAβgeobpA (column 8 to 13) as probe, which reveals the floxed sequences and the pBR322 sequences of the Cre transgene. Columns 1 to 3 were loaded with the DNA of a floxed double transgenic progenitor Sycpl -Cre / βgeo (1 represents the liver, 2 the kidneys and 3 the testes). Columns 4 to 6 correspond to the DNA extracted from the tails of three babies obtained with a normal mouse. Column 7 corresponds to the DNA of a mouse carrying the Sycpl -Cre trangene (control). Column 8 represents the DNA and column 9 the DNA of the testes of a fluxed Sucpl / Cre Pgkl double transgenic progenitor. Columns 10 to 13 correspond to the DNA of the tails of four babies obtained. Restriction fragments are indicated by "c" which is the fragment of Cre transgene; "in" which represents the internal fragments of the transgenes predicted from their nucleotide sequences (the respective sizes are 3.9 and 2.4 kb for βgeo and 3 for Pgkl); "j" corresponds to the junction fragments with the neighboring cell sequences. The transgene is represented by a single line, the chromosomal sequence by a double line, and the LoxP sites by a triangle.

Detailed description.

The original method of the present invention therefore makes it possible to transfer a recombinase into the oocytes in order to rapidly and with high efficiency perform all site-specific recombination operations in the mammalian genome, in particular the insertion of a foreign sequence (knock in), sequence replacement or gene silencing, and meiotic recombination between parental chromosomes (See Figures 1 and 2). This method is based on the surprising demonstration that the recombinase, expressed during meiosis, is transmitted in association with the chromatin of the sperm and is therefore capable of catalyzing a recombination of predefined site in the embryo before the first division.

It should be noted that the Cre transgene is present in the father in the hemizygous state. This means that only 50% of the descendants receive it. On the other hand, all receive the enzyme associated with the gamete's chromatin, and the recombination takes place effectively and only in embryos at the 1 cell stage, which ensures the subsequent stability of the recombined LoxP sites.

In addition, if the transgene is transmitted, the activity of the promoter is extinguished in somatic cells throughout the life of the organism, from embryonic development to its senescence. Thus, the genetic modifications carried out are reproduced during development in all the cells of the organism, without detectable chimerism, said modifications remaining stable and immediately hereditary. The method presented is applicable to organisms for which transgenesis techniques have so far been difficult to develop (absence of ES cell lines).

Transfer of Cre recombinase by sperm chromatin. In the case of the Tcp-10-bt-Cre transgene first, then in that of the Sycpl -Cre transgene, it has been shown that Cre enzyme molecules synthesized during spermatogenesis remain associated with the compacted chromatin of the spermatozoon. Inactive in this context, they can nevertheless carry out recombination between LoxP sites when returning to an unpacked nuclear structure. This operation, which can be performed on sperm under experimental conditions, takes place naturally after fertilization in the male pronucleus of the zygote.

The recombinase activity was tested in the following experiments:

In a first series of experiments, fertilized eggs were obtained from wild type females crossed with hemizygous males for the Tcp-lObt-cre transgene and for a multicopy insertion of the floxed coding region of the βgeo gene without promoter. A construction with a LoxP site located 3 ′ of the Pgkl promoter was microinjected (FIG. 3A). according to standard techniques (Hogan, 1986). After 24 hours of culture, approximately one in four embryos reveals a positive staining with X-Gal (FIG. 3B, C, and D). The expression of the βgeo gene is due to the efficient integration of the promoter microinjected upstream of the coding region.

A simpler genetic configuration such as the structure Rxrα ^{ΛAF2 (L)} was used in order to demonstrate the specific site integration of the microinjected DNA.

This second series of experiments demonstrated the presence of latent Cre activity in the spermatozoa of the Sycpl -Cre foreign mice, in which the excision of floxed sequences takes place during gametogenesis leaving only a single LoxP site at the time of fertilization. . In addition, these experiments made it possible to know whether the recombinase is still active in the male pronucleus and could allow the integration of a floxed transgene into a predetermined LoxP site. Males which have the Sycp-1-Cre transgene and the ^floxed Rxrα ^{AAF2 (LNL)} locus were obtained (Figure 4A). In the sperm of these males, the tkneo sequences were abundantly excised to generate the recombinant allele Rxrα ^{ΔAF2 (L.} This allele is transmitted to the whole progeny which only keeps one remaining LoxP site. The double transgenic mice were crossed with wild type females; the embryos fertilizers were isolated; and a recombinant DNA comprising the coding sequence for the protein for the GFP protein (Green Fluroescent Protein) upstream of a splice acceptor and an IRES (Internai Ribosomal Entry Sequence) (FIG. 4B) a PCR analysis of the genomic structure, shown in Figure 4D, shows that the coding sequence for GFP has been inserted into the Rxrcc ^ ² ^ locus (Figure 4C) for many babies and that none animal keeps the LoxP locus intact Animals of the Rxrα + / + type are under-represented.

The locus RXRa-LoxP-GFP-LoxP resulting from the recombination is transmitted and found in somatic tissues of the offspring of females and males Cre ^". The expression of GFP was demonstrated by fluorescence microscopy in the skin and Among the multiple applications offered by the efficient transfer of a recombinase by sperm chromatin, reporter mice, each having a genomic locus carrying an insertion with a LoxP site and a coding region for a reporter protein ( GFP, Iuciferase, β-galactosidase) can be obtained.

The introduction of a transgene in a single microinjection step can be used to identify a promoter, or to analyze a mutant promoter in a known genomic context and in all the organs of the mouse. Likewise, it is possible to insert various reporter sequences, mutated coding regions, genes coding for toxins, and any coding region of interest under the control of a given promoter.

The method for the targeted insertion of a sequence carrying a LoxP site microinjected into the fertilized egg produced from the crossing of a double transgenic male Sycpl -Cre / LoxP is presented in FIG. 4 and is summarized below: 1) The constitution by crossing of a double transgenic mouse carrying the transgene Sycpl -Cre and a structure in which two LoxP sites surround a foreign sequence (tkneo gene);

2) the two transgenes are kept intact in the somatic tissues of the mouse; synthesis of Cre in the germ line results in the excision of the floxed sequence (tkneo) in the sperm of the males;

3) a male is crossed with a wild type mouse for the Rxrα and Cre locus; the enzyme Cre remains associated with the LoxP site of the Rxrα allele mutated in chromatin (50% of embryos); independently 50% of the embryos receive the Cre transgene;

4) the recombinase is released after decondensation of the chromatin;

5) a recombinant DNA LoxP-SA-IRES-GFP-polyA is microinjected into the male pronucleus 15 hours after fertilization by conventional techniques (Hogan et al 1986);

6) all the embryos, Cre ⁺ or Cre ^" , having received the gene comprising a LoxP site, have inserted there the region coding for the protein GFP by virtue of the action of recombinase;

7) expression: the LoxP site had been inserted into an intron of the Rxrα gene. The presence of a splice acceptor (SA) allows the inclusion of the sequence

IRES-GFP in a functional messenger, that of an IRES site (Internai Ribosome Entry Site) allows the translation of RNA whose 3 'end is defined by the poly-adenylation signal ("polyA") added downstream of the coding region. The green fluorescence characteristic of the expression of the new reporter is demonstrated in the organs known to express the Rxrα receptor. In the experiments carried out during the accomplishment of the present invention, the recombination efficiency was between 50 and 80% (recombinants related to the number of mice born after microinjection).

It goes without saying that the process of the present invention, in particular illustrated above, is in no way limited to a particular region for the insertion of the transgene, to a particular transgene, to a particular promoter controlling the expression of a site-specific recombinase, or alternatively to a specific site-specific recombinase.

The range of applications made possible by the transfer of a recombinase by the chromatin of the spermatozoa is notably illustrated by the following different cases:

Identification of a tissue-specific promoter.

A "reporter mouse" is produced which carries a transgene comprising the LoxP-IRES-GFP-polyA sequences integrated into a chromosomal site, either by transgenesis and illegitimate recombination, or by homologous recombination. The insertion upstream of cloned genomic fragments into a vector also comprising a LoxP site makes it possible, from the first generation of mice, to identify the animals, therefore the fragment which express GFP in the tissue of interest. Several "reporter mice" can be used in parallel, with different reporter genes (in particular β galactosidase or Iuciferase), at different chromosomal locations. It is also possible to insert a gene of interest coding for a protein of interest expressed, for example, in mammalian milk.

Analysis of a promoter's expression pattern.

The insertion of a reporter downstream of a promoter (GFP downstream of Rxrα) makes it possible to precisely establish the expression pattern of the intact promoter in its natural genomic location. Expression of heterologous genes ("knock in").

Depending on the expression pattern after the insertion of a reporter gene, the expression of any biologically active gene can be carried out under the transcriptional control of the promoter of interest. For example, one can insert a gene coding for a toxin, in particular the diphtheria toxin, under the control of a specific tissue promoter, and thus cause the destruction of given cells possibly at a given stage of development.

Mutant analysis.

The insertion of cassettes comprising different mutants of the same sequence makes it possible to compare their properties in a constant chromosomal context, and in constant or variable physiological conditions.

The Scypl promoter can be used in the process according to the invention.

The Sycpl gene is expressed only during the prophase of the first meiotic division in male and female germ cells (Meuwissen et al,

1992; Heyting et al. 1989; Offenberg et al. 1991; Dietrich et al. 1992; Moens et al.

1992; Dobson et al. 1994 and Dietrich et al. 1983). The nucleotide sequence of the 5 'region of the mouse Sycpl gene has been described by Sage et al. 1997 (Figure 5 A). The region upstream of the gene does not contain a TATA box, but the transcription site includes the initiator element CCA ^{+ I} GTCC (Smale et al. 1989). The screening of a genomic library with a Pstl fragment of the rat cDNA (692 bp) which covers the

585 bp from the 5 'end of the coding region of Sycpl and a piece of the untranslated leader sequence (Meuwissen et al. 1992; Sage et al. 1995), led to the identification of five genomic clones, of which l 'one of them includes the 5' region upstream of the transcription initiation site. The 5 ′ longest BamHl fragment which hybridizes with the 692 bp cDNA probe has been subcloned and its nucleotide sequence has been established. The nucleotide sequence in the region analyzed, extending from positions -324 to +268 shows an overall identity of 80% between the two species (Figure 5b).

The high degree of conservation between the two species is a clear indication that this region contains important regulatory elements.

The transgenic analysis and several series of deletions inside the Sycpl promoter made it possible to define two different functional regions. A strong enhancer is present between positions -260 and -54. A proximal element 59 bp long (-54 to

+5), is sufficient to direct the expression of heterologous reporter genes (lacZ and read) in the appropriate male germ cells (Figure 5).

Since the enhancer present in the Sycpl promoter is active in cell lines of various origins, it is clear that this promoter depends on the action of ubiquitous transcription factors. The fragment between positions -54 and +5 is sufficient to direct expression of the Iuciferase in primary spermatocytes. Sequence comparisons between the four early meiotic genes in mice (Sycpl, Hsp70-2, Pdha-2, and Xmr), revealed no conservation of a binding site for transcription factors. However, there is a high degree of similarity (80%) between the sequences of the Sycpl promoter in mice and rats. For example, an E-box and a potential site for Myb-type transcription factors are kept.

In the ovaries, where meiosis is initiated during the early stages of development, no expression of the reporter genes is observed in the oocytes. Thus, the Sycpl promoter is very advantageous as a tool for directing the expression of Cre recombinase, because its activity profile allows the genetic stability of organisms and their descendants. Cre-induced recombination at the LoxP site has been programmed to occur during meiosis in male germ cells. The Cre gene has been expressed under the transcriptional control of a promoter region of the Sycpl gene, which is active at leptotene-zygotene stage of meiosis in males. Despite the fact that the gene is expressed in male and female gonads, the selected region of the promoter which was used in the construction Sycpl -Cre led to the expression of the transgene exclusively in the testes. As a result, flox sites have never been altered in the progeny of females. Floxidized sequences were maintained stably in somatic tissues (except in the testes. No expression was detected during the embryonic stages post-implantation and in adult tissues even using the very sensitive RT-PCR technique (Meuwissen et al. 1992) Cre-induced recombination in the testes represents an advantageous process compared to that described in previous studies on the activity of recombinase in other tissues of the mouse (Akagi et al. 1997; Gu et al. 1994; Wagner et al. 1997) Indeed, when we test the progeny obtained by the process of the present invention, we see that all the floxed alleles transmitted by males have undergone recombination. Such a recombination frequency is particularly suitable for the Cre / lox recombination system.

Cre recombinase can be expressed at the early stage of spermatogenesis.

The promoter from the Sycpl gene (Synaptonemal Complex Protein 1), isolated exclusively during the early stages of meiosis, was isolated and target expression obtained during the zygotene to pachytene stages of reporter genes in transgenic mice. This promoter, as well as that of a later meiotic promoter (Tcp-10-bt, Ewunolu et al. 1993), was used to express the CRE enzyme during spermatogenesis in transgenic mice. In the case of Sycpl, we observe in males who also carry a floxed transgene, the excision of these sequences during spermatogenesis. This excision does not take place in males carrying the TcplO-bt-Cre transgene. The recombinase is well synthesized, but at a period when the restructuring and compacting of the chromatin begins, which is very probably the cause of the absence of recombination before the formation of the spermatozoon. Recombination of LoxP sites during meiosis.

It has been shown that expression from the Sycpl -Cre transgene during the pachytene stage of the first meiosis division allows recombination between LoxP sites at non-identical positions on the chromosomes of paternal and maternal origin, according to the scheme of FIG. 2. The molecular markers available on the paternal and maternal chromosomes make it possible to show an exchange between chromatids paired at the LoxP sites. The frequency of the recombination event in the offspring of Sycpl -Cre males carrying the two floxed chromosomes is of the order of 30%. From this example, any construction of recombinant chromosomes is possible from LoxP sites introduced by conventional methods of homologous recombination into the ES cell, from sites inserted by transgenesis and illegitimate recombination, or from sites introduced by addition methods. targeted.

Activity of the Sycpl promoter in various cell lines.

A series of recombinant DNAs was constructed with different parts of the 5 ′ sequence upstream of the transcribed region of Sycpl (FIG. 5a) linked either to the gene coding for β-galactosidase or for Iuciferase. First, a search was made for cell lines which express the Sycpl gene in order to obtain an experimental basis for the identification of the genetic elements important for expression during meiosis.

A series of non-germinal cell lines were then used to measure the expression of reporter genes after transfection of the recombinant DNAs, in the hope that at least low activity could reveal the presence of the promoter. Unexpectedly, all of these cell lines showed a high level of luciferase activity after transfection of the various recombinant DNAs (FIG. 6). These results demonstrate that even the smallest Sycpl fragment extending only up to nucleotide -54 allows the expression of Iuciferase (at a low but detectable level). A distinct activity of the promoter, although not related to a specific activity of meiosis, is therefore controlled by the sequences of the 5 'region close to the Sycpl gene. We can therefore conclude that either silencer elements not included in the longest fragment tested (1950 bp) prevents the expression of Sycpl in somatic cells in vivo, or the germline specificity depends on a signal system lost in the cells somatic in culture.

Specific expression in the testes of transgenic mice

Four different fragments of the Sycpl promoter were tested for their capacity to direct the expression of reporter gene in transgenic mice ([- 1848 / + 102], (-722 / + 102], [-260 / + 102] and [-54 / + 102]).

The three longest promoter fragments were tested based on expression of the lacZ gene. Knowing that the low level of expression by the fragment [- 54 / + 102] and that the background noise of the β-galactosidase activity in the testis extracts is relatively high (Shaper et al. 1994) the Iuciferase was used in this case as a reporter. By comparison, the expression of the Iuciferase was tested in parallel for the fragment [260 / + 102]. The results, presented in Table 2 below, demonstrate that, in each case, at least two families express the transgene exclusively in the testes. This was observed even for the mouse which carries the shortest Sycpl sequence [-54 / + 102].

TABLE 2

TRANSGENIC LINES COMPRISING FRAGMENTS OF THE PROMOTER Sycpl.

Transgenic name and Length of the Number of lines Number of adult mice Profile of the expression gene reporter transgenic fragment tested independent

[-1848 / + 102] lacZ 1950 2> 10 Spermatocyte specific

[-722 / + 102] lacZ 824 5> 50

[-260 / + 102] lacZ 362 2 (+31> 10

[-260 / + 102] luc 362 2 ( ⁺ l ^* )> 20

[-54 / + 102] luc 156 2 (+ 1 ")> 20

[-260 / + 5] luc 265 3 (+3 ^* )> 10

^* lines without expression lines with expression

However, the level of expression is lower in the families [-54 / + 102], which implies that the sequence extending to the nucleotide -54 exerts an enhancer effect in vivo, as is the case in the transfected somatic cell lines.

This quantitative effect was evaluated by comparing the enzymatic activities in the testes of mice carrying the reporter Iuciférase controlled either by the promoter [- 54 / + 102], or by the promoter [-260 / + 102] of Sycpl. The latter has consistently shown a level of activity 10 to 100 times higher (Figure 7).

Expression during meiosis in adult testes.

The specific expression of β-galactosidase in the testes was confirmed by X-gal staining of several organs. In order to identify the cell types expressing the transgenes, the β-galactosidase activity was then analyzed in situ in sections of the testes.

The X-gal positive cells of [-260 / + 102] lacZ animals correspond to the second layer of germ cells located at the periphery of the tubules. An over-staining with hematoxylin confirmed that the lacZ positive cells are part of the pachytene stage III-IV spermatocytes of the seminiferous epithelium cycle (Clermont et al. 1972). At the early zygotene and pachytene stage, a weaker β-galactosidase expression could be detected in certain sections. No cell staining was observed in the postmeiotic compartment of the tubules or for the early zygotene and diplotene spermatocytes.

Endogenous expression of the Sycpl gene is absent or very weak during these stages (Figure 8). These results indicate that the sequences involved in the induction or suppression of the expression of Sycpl are localized among the fragments of the promoter analyzed. In order to determine whether the part of the Sycpl promoter (+1 to +102) present in these fragments plays a role in establishing the expression profile of the reporter genes, two additional transgenic families were generated by microinjection of recombinant DNA. deleted from these fragments, but retaining the initiator of the transcription of Sycpl with the sequences upstream to nucleotide -260. The enzymatic experiments demonstrate that the specificity and the level of expression of the promoter [-260 / + 5] are comparable to those of the promoter [- 260 / + 102] in the transfected cells (FIG. 6) and in the transgenic animals (FIGS. 9 and 10, compared to Figure 5).

Regulation during development in prepubertal mice.

The expression of the reporter genes in immature animals was measured in order to demonstrate that the expression of a transgene is identical to that of the endogenous Sycpl gene. The first wave of meiosis in the juvenile testes is synchronous with the appearance of the first meiotic cells, leptotene spermatocytes, about 10 days after birth.

The first pachytene cells appeared in the \ ^4th day and the first haploid cells after three weeks (Bellvé et al. 1977). We have analyzed the transgenic families [-260 / + 102] lacZ, [-260 / + 102] luc, [-260 / + 5] luc, and [-54 / + 102] luc.

The in situ analysis (FIG. 8) made it possible to detect the expression of β-galactosidase in the primary spermatocytes for the mouse [-260 / + 102] lacZ at the 13 ^th and 15 ^th day after birth, but not at the 8 ^th day. In the 10 ^th day, the expression was detected only in some mice, and in cases where the result is positive, it was observed that in some tubules. These results were confirmed by the measurements carried out on transgenic mice [-260 / + 102] luc. No expression of luciferase was detected before 10 ^ee day after birth. In the [-54 / + 102] luc and [-260 / + 5] luc mice, the time profile of the luciferase activity is the same, is identical to that of the endogenous gene (Sage et al. 1997).

All of these results made it possible to conclude that a fragment of 59 bp (- 54 / + 5) of the promoter Sycpl, covers an initiator sequence of 7 bp and a fragment immediately upstream of 52 bp which is sufficient to direct the expression of a transgene at the start of the first meiotic division in the male gonads.

None of the transgenes that are expressed in the testes are transcribed in the ovary.

In mammals, female gametogenesis takes place during embryonic development and is stopped before birth at the dictyotene stage. During prophase of the first meiotic division, the synaptonemal complex is morphologically identical to that of spermatogenic cells (Dietrich et al. 1983, Scherthan et al. 1996); and is more visible at the pachytene stage (Dietrich et al. 1992). This phenomenon is observed in the fetal ovary during the 15 ^th and 11 ^th embryonic days (E15 and El 7). Immunolocation experiments carried out on cryosections with an antibody directed against the rat SCPl protein (Meuwissen et al. 1992; Dietrich et al. 1992) made it possible to detect the SCPl protein (coded by Sycpl) in the ovaries of the mouse El 6 and El 7. The expression in the ovaries of reporter genes expressed by various transgenic families was then analyzed during male meiosis. Neither the in situ detection of β-galactosidase nor the more sensitive Iuciferase test made it possible to detect expression of the transgene. RT-PCR experiments carried out with RNA from the El 6 ovaries of [-

1848 / + 102] lacZ and El 7 ovaries of [261 / + 102] lacZ transgenic mice failed to detect the transcripts fused between the first Sycpl exon and the lacZ sequence, while expression of the endogenous gene was detected in the same samples (Figure 11). One possible explanation for the absence of expression is the use of an alternative Sycpl promoter in the female germ line, as was shown for the DNA methyltransferase gene (Mertineit et al. 1998).

To confirm this hypothesis, the 5 'RACE technique was used to determine the site of initiation of Sycpl transcription in meiotic cells originating from the El 6 ovaries. Twelve clones containing the longest cDNA fragments among 48 clones analyzed, have been sequenced. These sequences clearly indicate that the transcription of the transcription initiation site in female germ cells is identical to that previously defined in male germ cells. It thus appears that the signals which ensure the temporal and spatial expression of the Sycpl gene during male meiosis are not sufficient for its expression in the female.

Somatic maintenance of floxidized sequences in the tissues of transgenic Sycpl -Cre / LoxP males.

Four independent transgenic families carrying the transgene have been established

Sycpl -Cre. No difference in the properties of these families having been noted, we randomly chose a family for the rest of the studies. The results obtained demonstrated that the stably maintained transgene was only transcribed in the testes.

Double transgenic lines, which maintain Sycpl -Cre with a floxed transgene were obtained by crossing a Sycpl -Cre mouse with a mouse whose genome carries a floxed transgene. Techniques for integrating a floxed transgene into the genome of a mammal are described in particular in Kuhn and Schwenk, 1997, Galli-Taliadoros et al. 1995, and Feil et al. 1996. In the present case, two target sites were created by microinjection inside the fertilized eggs of cloned DNA: a tandem repetition of a βgeo sequence flanked by LoxP site (Friedrich and Soriano, 1991) and a tandem insertion at low copy number (less than two copies) of an active promoter (LoxP -Pgkl). A third target site corresponds to the Rxrα ^{ΔAF2 (LNL)} allele (Feil et al. 1996), wherein a cassette LoxP-tk-neo-loxP has been inserted by homologous recombination into the 8 intron ^me RXRa. In males carrying LoxP-βgeo and LoxP-Pkgl transgenes, "Southern blot" hybridization indicates total stability of these loci in somatic organs.

A more detailed analysis carried out on Sycpl -Rxrα ^{ΛAF2 (LNL)} animals has shown that part of the males (2 out of 8 tested) maintain in their somatic DNA both recombinant and intact floxidized sequences. This observation is in agreement with the results of the studies on the Sycpl promoter mentioned above. Thus, the structure of floxed transgenes established at birth remains stable in all somatic organs, even in the case of senescent males. The chimeric animals were discarded and further studies were carried out with males in which the sensitive PCR test did not make it possible to detect somatic recombination. The modified floxed sequences were in each case only detected in the testes and in the sperm DNA.

Deletion of the floxed locus during transmission by Sycpl-Cre / LoxP males.

In the offspring of crosses between double transgenic animals with non-transgenic partners, excision of the intervening sequences have always been observed when the floxed locus was transmitted by the Sycpl - Cre / LoxP males, while the same loci were completely stable when they have been transmitted maternally. These results are shown in FIG. 12 for the offspring of a wild type female with a male heterozygous for the floxed allele Rxrα ^{ΔAF2 (LNL)} and hemizygous for the transgene Sycpl -Cre. PCR amplification of DNA extracted from the father's tail (column 1) did not detect the presence of floxed sequences (fragment called "tkneo"). None of the nine babies (columns 2 to 10) have this fragment. This result actually corresponds to two distinct situations:

- Two babies (columns 4 and 10) received the wild Rxrα allele from the father (identified by the characteristic amplification of the product ("Rxrα"). The other babies inherited the Rxrα ^{ΛAF2 LNL)} allele for which the tkneo sequences were excised.

Amplification with the two primers RXR1 and RXR2 generated a larger fragment characteristic of the recombined locus ("Rxrα ^{ΛAF2 ()} ). The excision was observed regardless of the presence (columns 2, 6, 8, 9) or l absence (columns 3, 4, 5, 1, 10) of the Sycpl -Cre transgene.

Similar results were obtained for the multimeric β-geo-LoxP transgene and the Pgkl-LoxP transgene (Figure 13). The excision in this case is highlighted by the disappearance of the bands which correspond on the "Southern blots" to the internal fragments predicted from the sequence of the transgene ("in"), while the fragments of the junctions with the endogenous sequences ("j") have been maintained in all cases. In the male parents, the floxed sequences were not modified (columns 1, 2, 8, FIG. 14), except for the DNA of the testes (columns 3 and 9).

Implementation of the process according to the invention with the promoter Tcp-lObt:

In the context of the invention, the promoter of one of the genes of the Tcp-lObt t locus was also used to direct the expression of the Cre recombinase in male germ cells. Those skilled in the art have access to all the information necessary in Ewunolu et al., Development, 1993 117: 89-95 on the characterization of the Tcp-lObt promoter in transgenic mice. Analysis of the expression of reporter genes (for example the gene coding for β-galactosidase) made it possible to conclude that this promoter makes it possible to mark the population of germ cells in the late phase of meiosis (late pachytene in the haploid stage) .

Targeted deletion of genes floxed by the Tcp-lObt- transgene has been performed

Cre. For this purpose, two independent lines carrying the Tcp-lObt-Cre transgene have been established. We first examined the activity of the Cre enzyme in three different types of "floxed" transgenic mice. Whatever the constructions, a gene without promoter (β-geo), a promoter which is not active in the testis (pgk-1) in multiple copies or the tk-neo cassette in single copy introduced by homologous recombination into the Rxrα locus, these remain completely intact in the male germ cells (including in the spermatozoa purified from epidydime).

On the other hand, the majority of the descendants obtained by these males excised the floxed fragment. Analysis of the recombination in purified sperm and the descendants of these males has shown the presence of the enzyme Cre inactive in these germ cells by the fact that the Cre recombinase regains its activity following the decompaction of the nucleus of the sperm. in the fertilized egg of the mouse or in a test tube by biochemical processes. This observation of the maintenance of the Cre protein associated with the compacted chromatin of the spermatozoon, of its transfer to the zygote, where it is active in the male pronucleus after fertilization, opened the way for the operation of inserting a foreign sequence. at a genomic LoxP site by simple microinjection into the fertilized egg. The relatively simple operation consists in establishing by crossing a male carrying two transgenes. One is Tcp-IObt-Cre, the second is at least a LoxP site at a chromosomal locus (if the locus contains two LoxP sequences, the floxed region will be eliminated during decompaction, reducing to the case of a single site) . The Cre enzyme will only be synthesized in the late stage of meiosis, but will be present in all haploid cells, the Cre gene itself being inherited by only 50% of gametes. The presence in the nucleus of the spermatozoon, and therefore in the pronucleus of the stage 1 embryo, of a chromosomal LoxP site and of the active recombinase, allows targeted integration of an exogenous DNA carrying a LoxP site introduced by microinjection. The efficiency of this integration was measured with two different combinations of insert and recipient locus: integration of a promoter (pgkl) upstream of a lacZ coding sequence of the c-myc promoter upstream of the same sequences. In both cases, the integration efficiency at the target site was equal to or greater than 50% of births.

Example 1: Cell culture and transfection experiments.

The BALB / 3T3 cell line (clone A31 American Type Culture Collection No. CCL-163) and was transfected with 2 μg of the chimeric promoter with 0.1 μg of a CMV-lacZ Control construction, using the calcium phosphate co-precipitation method (Sambrook et al. 1989). 2 x 10 ⁴ cells were inoculated per 35 mm dish. The enzymatic tests were carried out two days after the transfection.

Example 2: Microinjection and analysis of transgenic mice.

The transgenic mice were generated by microinjection of a plasmid DNA inside the fertilized eggs according to techniques commonly used by those skilled in the art (Hogan et al. 1986). Verification of the foreign animals was carried out by Southern and / or Dot blot analysis of the DNA end (Sambrook et al. 1989). All the transgenic families were generated and maintained in a C57BL / 6xDBA / 2 FI genetic background.

EXAMPLE 3 Isolation of the Genomic Clones of Rats

The rat genomic clones were isolated by screening a genomic library (Sambrook et al. 1989) in a lambda DASH vector (stratagene) with a rat cDNA primer (Meuwissen et al. 1992).

Example 4: Analysis of the sequence

DNA sequencing was performed using the Sequenase II kit

(Amersham). The Genetic Computer Group's FASTA computer program was used for sequence comparisons in databases. The accession numbers in the Genbank and EMBL databases for the promoter sequences in rats and mice are AF020295 and AF020296, respectively.

EXAMPLE 5 Plasmid Construction

All the fragments of the Sycpl promoter come from a genomic clone containing 12Kb of sequences upstream from the site of initiation of the transcription of Sycpl (FIG. 5) (Sage et al. 1997). At their 3 'end, all of the promoter fragments tested (with the exception of a fragment, see infra) extend to the SacII site which is located in the first exon at position +102 (Sage et al. 1995). Cleavage of the genomic clone with BamHI and SacII led to a 2Kb fragment which was then treated with the Klenow fragment of DNA polymerase (Biolabs) and inserted at the EcoRV site in the vector pBluscript KS (-) (stratagene). A 1950 bp EcoRI-Xhol fragment was then subcloned into the pnassβ plasmid (clontech, accession number U02433) carrying the lacZ reporter gene ([-1848 / + 102] lacZ plasmid). In order to obtain chimeras comprising shorter genomic sequences, PCR amplifications were carried out using internal oligonucleotides of 18 bp 5 ′ primer in conjunction with the T3 primer on the same BamHI-SacII fragment subcloned in pBluescript. The PCR fragments were cloned into the vector pBluescript KS (-), verified by sequencing and transferred to the EcoRI-Xhol sites of the plasmid pnassβ. The same promoter fragments were then transferred to the reporter gene Iuciferase in the plasmid pXPl (Lee et al. 1994) (these plasmids have the same names as before with "read" instead of "lacZ". An additional plasmid has was constructed with the 5 'sequences upstream between positions -260 and +5. A PCR fragment was amplified with the same 5' primer used for the chimera [-260 / + 102) lacZ, the other primer covers the site Sycpl transcription initiation (position + 5 / - 15). This 275 bp fragment was subcloned into pXPl to form the plasmid [-260 / + 5] luc.

Example 6: Isolation of TARN and Analysis

Total RNA was prepared according to the isothiocyanate method (Chomezynski et al. 1987). Control RNA and Myb ^ ^' A mouse samples were used (Toscani et al. 1997). Fetal ovaries from a pregnant female were collected, and in order to ensure that the RNA preparations are comparable, the same amount of testis and liver was used. The total RNA was treated with DNasel (Boehringer), then extracted with phenol and precipitated with ethanol. 2 μg of RNA was reverse transcribed and amplified using the Titan kit from Boringer. With regard to the control reactions, the RNA was pretreated with RnaseA. The sequences of the primers used to detect the Sycpl transcripts correspond to positions 115-135 and 495-475 in the cDNA (Sage et al. 1995), leading to a product 380 bp long. The oligonucleotides used to detect the fusion transcripts between them Sycpl and lacZ is found at position 50-68 in the cDNA of Sycpl and at position 444-425 in the lacZ reporter of the plasmid pnassβ. The conditions of the PCR cycles were 30 seconds at 94 ° C, one minute at 55 ° C and 30 seconds at 72 ° C for 32 cycles. The products of this reaction were hybridized with an internal probe (position 245-265 in the cDNA Sycpl and 358-378 in the plasmid pnassβ. The cDNA fragments Sycpl -lacZ were subcloned in pBluescript and sequenced in order to confirm the correct splicing of the SV40 intron.

The 5 'end of the Sycpl gene in oocytes was determined using the 5' RACE kit from Boehringer following the procedure previously described in Sage et al. 1997.

EXAMPLE 7 Test of the Activity of the Reporter Genes

Β galactosidase activity was tested on tissue extracts using the Galacton substrate as described in Shaper et al. 1994. The in situ determination was carried out using an alternative procedure to cryosection, which allowed better conservation of the structure of the seminiferous epithelium. The albuginea was removed and the tissue was carefully stretched with the help of forceps to facilitate penetration of X-gal. A fixation in 2% of paraformaldehyde for one hour at 4 ° C precedes an incubation for 16 hours at room temperature in 0.2% of X-gal. After staining, the testes were post-fixed for 24 hours in 10% paraformaldehyde and inclusion was carried out in the "Leica Historesin" following the manufacturer's instructions (Leica Instruments). Sections 5 μm thick were over-colored with Harris hematoxylin. Iuciferase activity was measured using the "Luciferase Asay System" (Promega) according to the manufacturer's instructions.

EXAMPLE 8 Munolocation of the Mouse SCPl Protein

Cryosection 8 μm thick from embryonic testes aged 15 or

16 days (E15 or E16) were incubated with polyclonal antibodies directed against the rat protein as previously described in Meuwissen et al. Examples 9: Construction of the Plasmids and Production of the Transgenic Mice.

The Sycpl -Cre plasmid was constructed in two stages. A fragment of the promoter [-722 / + 102] of the Sycpl gene extending from -722 to +102 relative to the site of initiation of transcription (Sage et al. 1997, see above) was transferred into the plasmid pBluescript KS (Stratagene) between the EcoRI and Xhol sites. The coding sequence for the Cre recombinase (Genebank accession number XO3453) and a polyadenylation signal was then inserted into the first plasmid digested with the restriction enzymes Sali and Kpnl. In the plasmid pβgeo-LoxP (Friedrich and Soriano

1991), the βgeo fusion gene was integrated between two LoxP site sites. This plasmid was constructed by inserting a HindIII-BamHI fragment corresponding to the coding sequence originating from the plasmid pβgeo inside the HindII-BamHI sites of a plasmid pBS112 containing two LoxP sites (Friedrich and Soriano 1991). In the plasmid pPGK-LoxP, the promoter of the mouse Pgkl gene (Accession number

Genebank M18735) is inserted between two LoxP sites. This plasmid was constructed by inserting the Pgkl promoter inside the HindIII-BamHI sites of pBSl 12.

EXAMPLES 10 The Transgenic Mice

The transgenic mice were obtained by microinjection of the chimeras linearized by Pvul inside the fertilized eggs according to techniques well known to those skilled in the art (Hogan et al. 1986). The DNA was purified on Qiagen columns in accordance with the manufacturer's instructions, then linearized, and suspended at a concentration of 5 ng / μl in sterile water before microinjection. Verification of the transgenic animals was carried out by Southern and / or dot blot analysis of the DNA extracted from the tail of the mice. All the transgenic families were generated and maintained in a C57BL / 6 x DBA / 2 FI genetic background. RXRalpha the mutant N ² O ⁴ has a cassette LoxP-tk-neo-loxP, inserted by homologous recombination inside the 8 ^th intron of the gene RXRalpha (Feil et al. 1996). Example 11 Expression of the Cre Transgenes

The level of Cre mRNA was estimated by reverse transcriptase analysis (RT-PCR). Isolation of RNA from the brain, kidney liver and testes was performed using phenol acid extraction. The total RNA preparations were treated with DNasel (purified of any residual RNase) and the cDNA was synthesized for 30 minutes at 50 ° C using 50 IU of the reverse transcriptase of the murine Moloney leukemia virus ( Boehringer Mannheim) using 1 μg of RNA as a template. It was then amplified for 35 PCR cycles using the primers Crel (5'-TGA TGG ACA TGT TCA GGG ATC-3 ') (SEQ ID n ° 4) and Cre2 (5'- CAG CCA CCA GCT TGC ATG A -3 ') (SEQ ID No. 5) constituting a fragment of the Cre cDNA of 865 bp.

Example 12: Genotyping of the Mouse

The end of the mouse DNA was prepared as described in Hogan et al. 1986 and analyzed by slot blot hybridization using a series of probes. The Cre probe can be obtained by PCR amplification of any plasmid which contains the open complete reading phase for the Cre protein using the primers Crel and Cre2. All these fragments were purified twice on agarose gel. Probes can also be prepared from the sequences of the "floxed" transgene.

Example 13: Detection of excision induced by Cre

Southern blot analysis was carried out according to techniques well known to those skilled in the art (Sambrook et al, 1989). The DNA was digested overnight with the restriction enzymes indicated above (ten times in excess). The DNA fragments were then subjected to electrophoresis and transferred to nitrocellulose. The hybridizations were carried out using the radiolabelled pSAβgeo or pPKβgeobpa plasmids (Soriano et al, 1991). In addition to the "floxed transgenes", the sequences of the plasmid pBr322 present in these chimeras allow the detection of the Cre transgene. The analysis of the double transgenic mice carrying the expression vector Cre and the allele Rxrα ^{ΔAF2 LN)} was carried out by PCR amplification as described in Feil et al. 1996. With regard to the detection of the Rxrα ^{ΔAF2 (LNL)} allele, the floxed tkneo sequences were amplified using the primers 5 'and 3', 5'- GGT TCT CCG GCC GCT TGG GT- 3 '(SEQ ID # 6) and 5'- GGA GGC GAT GCG CTG CGA AT-3 '(SEQ ID # 7), respectively. As regards the detection of the Cre transgene, the primers Crel and Cre2 were used. The visualization of the excision of the floxed tkneo marker of the target allele Rxrα ^{AAF2 (I} ^ ⁾ was used using the primers RXR1 (5'- CCA GGA GCC TCC TTT CTC CA-3 ') (SEQ ID No. 8 ) and RXR2 (5'- CCT GCT CTA CCT GGT GAC TT-3 ') (SEQ ID n ° 9). These primers amplified a fragment of 156 bp from the wild type Rxrα allele and a fragment of 190 bp from the recombinant allele Rxrα ^{ΔAF2 L)} . The amplified products were analyzed by electrophoresis on 2% agarose gel.

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Claims

1. A method of inserting a foreign sequence into the genome of an animal comprising the following steps: a) Expression of a site-specific recombinase in the germ cells of a male carrying at least one specific site in his genome , said recombinase remaining associated with the chromatin of the spermatozoa. b) Fertilization of the oocytes and transfer of the recombinase by said spermatozoa. c) microinjection of a recombinant DNA having the structure “specific site- foreign sequence” in the male pronucleus after fertilization, d) insertion of said foreign sequence at the specific site in the male pronucleus of the 1 cell stage embryo, catalyzed by the recombinase released during the decondensation of sperm chromatin.

2. Method according to claim 1 characterized in that said foreign sequence comprises a gene of interest, to be expressed under the control of a promoter possibly tissue (s) specific (s).

3. Method according to claim 2 characterized in that said foreign sequence comprises a reporter gene fused to a promoter of interest.

4. Method according to one of claims 1 to 3 characterized in that said foreign sequence further comprises a splice acceptor element (SA), an IRES sequence and / or a polyadenylation signal.

5. Method for replacing a sequence X by a sequence Y in the genome of an animal characterized in that it comprises the following steps: a) Expression of a site-specific recombinase in the germ cells of a male carrying a structure “specific site-sequence X-specific site” in its genome, said recombinase remaining associated with the chromatin of the spermatozoa. b) Excision of the X sequence during spermatogenesis by recombinase. c) Fertilization of oocytes and transfer of recombinase by said sperm. d) Microinjection of a recombinant DNA having the structure “specific site- sequence Y-specific site” into the male pronucleus after fertilization. e) Insertion of the sequence Y at the specific site in the genome of the embryo at stage 1 cell, catalyzed by the recombinase released during the decondensation of the chromatin of the spermatozoon.

6. Method according to claim 5 characterized in that it allows the inactivation of a gene of sequence X.

7. Method according to one of claims 1 to 6 characterized in that said recombinase is Cre and said specific site is a LoxP site, or a variant or mutant of the LoxP sequence.

8. Method for transferring Cre in its active form into an oocyte, characterized in that it comprises the following steps: a) Expression of Cre in male germ cells comprising in their genome at least one LoxP site, said protein remaining associated with sperm chromatin. b) Fertilization of oocytes and transfer of protein by said sperm. c) Release of the protein in its active form during the decondensation of the chromatin after fertilization.

9. Method according to one of claims 1 to 8 characterized in that said recombinase is expressed under the control of a promoter allowing efficient expression in the spermatocytes of a mammal.

10. Method according to claim 9 characterized in that said promoter comprises at least one region of the promoter Sycpl, EF-1 alpha, IAP, FRMl ^, Pdha-2, Hsp70-2, Camll, Hit, β4-Gtase, Z / yl, Tcp-lObt, Pgk-2, Hst70, Hox-a.4 ^y , Proacrosin, c-Kit, ACE or Prml.

11. The method of claim 10 characterized in that said promoter comprises at least one sequence having at least 80% identity the region of the Sycpl promoter of sequence SEQ ED No. 1.

12. Transgenic animal capable of being obtained by the process according to one of claims 1 to 11.

13. Nucleotide sequence making it possible to effectively promote the expression of a gene of interest during the first meiotic phase of the spermatocytes of a mammal consisting of a sequence having at least 80% identity with the sequence SEQ ID No. 1.

14. Nucleotide sequence according to claim 13 which consists of the sequence SEQ ID No. 1 or SEQ ID No. 2.

15. Nucleotide sequence according to one of claims 13 or 14 fused to a coding sequence for a gene of interest.

16. Nucleotide sequence allowing the expression of a site-specific recombinase during the first meiotic phase of the spermatocytes of a mammal comprising a sequence having at least 80% identity with the sequence SEQ ID n ° 1 fused with the coding sequence for said recombinase.

17. Nucleotide sequence according to claim 16 characterized in that said recombinase is Cre.

18. DNA molecule comprising at least one sequence according to one of claims 16 or 17 characterized in that it is in the form of a plasmid, a viral vector, a pseudo-vector, or linear or circular naked DNA.

19. Transgenic animal containing in its genome a nucleotide sequence according to one of claims 13 to 17 allowing the expression of a gene of interest specifically in spermatocytes.

20. Animal according to claim 19 characterized in that the gene of interest is a site-specific recombinase.

21. Animal according to claim 20 characterized in that the recombinase is Cre.

22. Animal according to one of claims 20 or 21 characterized in that the recombinase remains associated with the chromatin of the spermatozoa.

23. Use of an animal according to claim 22 in the method of claims 1 to 11.

24. A method of recombination between paternal and maternal chromosomes characterized in that it comprises the following stages: a) Expression of a site-specific recombinase in the germ cells of a male carrying in its genome one or more site (s) specific (s), b) a said male is crossed with a female animal carrying in its genome one or more specific site (s), said recombinase catalyzing the recombination between specific sites during the first phase of meiosis.

25. Animal capable of being obtained by the method according to claim 24.

26. Multi-transgenic animal obtained by crossing animals between them according to one of claims 12 or 25.

27. Transgenic animal according to one of claims 12, 25 and 26 characterized in that it is a mammal, in particular selected from sheep, pigs, cattle, rodents.

28. Use of an animal according to claim 27 for the preparation of substances useful in medicine, in cosmetics, and / or in food industry, in particular of active substances, preferably peptides or polypeptides, antibodies, antigens, hormones, or growth factors, and / or for the manufacture of food supplements.

29. Use of an animal according to claim 27 as an experimental model for the identification of genes, promoter, proteins, especially genes involved in genetic diseases, and / or to test the action of active substances.

30. Use of an animal according to claim 27 for the preparation of organs or cells having better immunocompatibility with humans than wild type organs or cells or capable of having a low rate of rejection by the immune defenses of humans.

31. Use of an animal according to claim 27 for breeding.