CA2340594A1 - 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 TJENOME OF AN ANIMAL
BY ZYGOTIC TRANSFER OF A SITE SPECIFIC RECOMBINASE.
The present invention relates to a method for transferring into an oocyte of a mammalian site-specific recombinase, especially Cre, under its active form.
Said recombinase is expressed under control; a specific promoter in male germ cells with at least one specific site and remains associated with the sperm chromatin. This process is particularly useful for reshape the lo genome of an animal, for example to insert a foreign sequence in the genome of an animal, and to replace one sequence with another. In the case where said pronator is the Sycpl promoter, an application mode allows recombinations between paternal and maternal chromosomes. The present invention door also on animals likely to be obtained by this process, and on use ls of these transgenic animals in the pharmaceutical industries, cosmetics, and agro-food.
The site specific recombination process represents a powerful tool for carry out predetermined changes in the animal genome. Several enzymes of 2o recombination, including the Cre recombinase of bacteriophage P1 (Sauer &
Henderson, 1989, 1990 and Porter, 1998) catalyze a recombination event between of them defined double-stranded deoxyribonucleotide sequences (LoxP target sequence for the enzyme Cre). These Cre recombination operations were carried out in of cells in culture (Sauer and Henderson, 1990) and in cells of plants (Albert and 2s al. 1995), but not on the germ line of an animal.
The recombination reaction was most often used for the deletion targeted "knack-out" of specific genomic regions known as "floxed" by intramolecular recombination between two LoxP sites of the same molecule. In 3o in particular, deletions in specific cell types have been made from various genetic constructions allowing to express specifically the Cre recombinase (Brocard et al. 1997). Targeted "knock-in" integration could

2 represent a possible application of site-specific recombination and would be of a great interest, but certain limitations did not allow such development. Of in general, a recombinase could catalyze a recombination intermolecular between two DNA molecules each carrying a specific site (or s recombination site) and would be used for the integration of heterologous DNA
(introduced in the cell) at a defined site (a region of the cell's genome).
Application to the germ line of a mammal could be considered in mammals by currently available methods but the operation would require lo a series of long technically difficult stages, involving the selection of clones recombinants in ES cells in culture, Poltention of chimeras germinal, and a series of crosses before obtaining a stable recombinant line. Of a share the time required for all of these operations would be in months, otherwise in years and the yield would be low. On the other hand, the selection of recombinant clones would require 15 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 recombination homologous or excision are frequently lethal. for animals. Thereby, the effect of 2o these mutations in the male germline cannot be evaluated since the process cyclical spermatogenesis involves a number of regulatory genes which exercise critical functions in other organs. An example of this situation has been described for the Brcal gene (Gowen et al. 1996, Zabludoi ~ 'et al. 1996).
limitation may be overcome by establishing spatial and temporal control of the expression of the 25 recombinase. The use of specific tissue promoters and promoters inducible, the use of adenoviral vectors and forms of the recombinase activated by hormones have been considered to overcome this problem (Akagi et al.
1997, Brocard et al. 1997, Gu et al. 1994, Kühn e; t al. 1995, Porter, 1998, Wagner et al.
1997).
These problems are solved by the present invention since recombinase, is only expressed in germ cells rr ~ âles. So unexpected, she stays

3 associated with sperm chromatin and is transferred by sperm in the ooeytes in active form after fertilization. This ultimately allows of reshape the genome.
An additional application could be meiotic recombination site-specific counterpart between paternal and maternal chromosomes. This would require the presence of the enzyme during the first division of meiosis (essentially at pachytene stage}, which is precisely the case in the experiments leading to the current invention.
Meiosis reduces the size of the genome and generates genetic diversity by homologous recombination, while maintaining genetic integrity by a process of repair. Little knowledge exists about the processes of regulation transcriptional in the early stages of meiosis. However, studies with 1 transgenic mice have recently led to the identification of promoters specific male cells; for example HSP70-2 (Dix et al. 1996), Pdha-2 (Iannello and al.
1997}, Pgk2 (Robinson et al. 1989), Tcp-lObt (Ewulonu et al. 1993) and Hox-a.4 (Berhinger, 1993).
2o Addressing the expression of Cre towards 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, none Creep zygotic transfer was observed. However, in the context of this invention we found that the transfer can be highlighted as soon as a site specific LoxP
2s is present in the sperm genome.
The Sycp1 gene has been identified, cloned and located on chromosome 3 of the mouse (Sage et al. 1997). Protein is a major component of the core of synaptonemal complex (Meuwissen et al. 1992). It is present exclusively 3o during the meiosis of the male and the female of the beginning of the zygotene stage to the stadium diplotene (Meuwissen et al. 1992, Heyting et al. 1989, Offenberg et al. 1991, Dietrich and al. 1992, Moens et al. 1992, Dobson et al. 1994), The concomitant expression of mRNA

WO OO / i2742 PCT / FR99 / 02053

4 and protein shows that the regulation of expression is at the level transcriptional (Meuwissen et al. 1992).
Unlike the promoters mentioned above, which are expressed to relatively late stages of erminal differentiation, Sycp i presents the interest of being expressed during the early phase of meiosis; spermatocytes. He was necessary identify and select the elements responsible for the expression specific of Sycpl. Since it was not possible to establish a cell line which expresses the gene endogenous, experiments using transgenic mice were carried out.
So, we have lü found that a region of the mouse Sycpl premotor, localized up to 2b0 bp in upstream of the Sycpl gene transcription initiation site, is not only sufficient to direct expression of a transgene during the initial period of the meiosis in the male, but is also the most effective in males without being active in (ovary. This Sycpl region is therefore useful for recombination meiotic between LoxP sites either carried on the same chromosome or at locations distinct from the paternal and maternal chromosomes.
All the operations necessary for the implementation of this invention requires only a series of microinjections and relocations, technical 2o routine today, on a limited number of fertilized eggs (one litter is the most often sufficient). Unlike an operation from cells ES, the procedure presented does not imply the use of selection systems, and time necessary 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 prior art documents describe, or suggest the present invention as defined below.

WO 00! 12742 PCT / FR99102053 Description Thus, the present invention relates to a method of inserting a sequence foreign in the genome of an animal comprising the following steps

5 a) expression of a site-specific recombinase in spermatocytes of a male carrying at least one specific site in a genome, said recombinase remaining associated with sperm chromatin.
b) Fertilization of oocytes and transfer of re; combinase by said sperm.
c) Microinjection of a Recombinant DNA Having the “Site” Structure specific Io foreign sequence "in 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 recombinase released during decondensation of sperm chromatin.
1s Advantageously, said recombinase is Cre and the specific site is a LoxP site.
In this method, said foreign sequence contains a gene of interest, â
express under control of a specific tissue promoter). The gene of interest can additionally have a splice acceptor element; age (SA), an IRES sequence (Interna!
Ribosome Entry Site) and / or a polyadenylation signal (e.g. AATAAA) and Ie 2o gene of interest can code for a protein of interest or be a gene reporter to identify specific tissue promoters.
In another aspect, the invention relates to a replacement method of a sequence X by a sequence Y in the genome of an animal which consists of cross a 2s male of genotype “specific site-sequence X- specific cite” whose spermatocytes express a site-specific recombinase, said recombinase remaining associated with the chromatin of the spermatozoa, then to introduce by micrainjection a molecule structure "specific site - sequence Y- site specific" in its genome, said recombinase catalyzing the excision of the X sequence during meiosis of 3o spermatocytes and integration of the Y sequence in the specific site just after oocyte fertilization. By sequence X or sequence Y, any what coding or non-coding sequence. This process can be used to inactivation of a X sequence gene.

6 According to the methods mentioned above and for the following description of the invention said recombinase can be Cre and said specific site a LoxP site and be expressed as ie control of a promoter allowing efficient expression in cells germinal s of a mammal. During the transfer, Cre remains; therefore associated with the chromatin.
Any Cre protein, its mutants, and variants, and any mutant and variant of the LoxP sequence, can be used in the context of present invention.
Cre, well known to a person skilled in the art, has been described in particular in EP 300 422 (page 4 lines S-33} and EP 220 009 (page 3 lines 31-39), incorporated in the description 10 by reference. Many plasmids containing the Cre sequence are there disclosed and have been deposited at the American Tissue Culture Collection such as BSY90 (accession number ATCC 53255) and pBS39 (ATCC accession number: 20772). The Cre sequence may be isolated from pBS39 by the restriction enzymes XhoI and SaII. Other systems Recombinase / site specific can be used as part of this invention.
is Mention may in particular be made of the FLP / FRT system derived from Saccharomyces cerevisiae, the S / RS system of Zygosaccharomyces rouxü and the Crin / gix system derived from bacteriophage Mu which are described in EP 814 165, incorporated in the description by reference.
2o The present invention also relates to an I> process for transferring into an oocyte a site-specific recombinase, in particular Cre, in its active form. This process consists in expressing recombinase in male germ cells possessing a strong affinity for its site of action on DNA, said protein remaining associated with the chromatin of the spermatozoa at its specific site (for example at its site LoxP
25 in the case of Cre), to fertilize oocytes and to transfer the protein by said sperm. The protein is then released in its active form during the decondensation of chromatin after fertilization.
Among the promoters that allow efficient expression in cells 3o germinal of a mammal, we notably retain the promoters Sycp1 (No.
access to 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. Through example, said promoter could comprise at least one sequence having at least of identity with the region -260 / + 5 (SEQ ID No. 1} of the promoter Sycpl of mouse. This WO 00/12742 PCTlFR99J02053

7 region is sufficient to effectively induce gene expression of interest specifically during early meiosis of the spermatocytes of a mammal.
Examples of promoters that can be used to accomplish the present The invention is illustrated in Table 1 below. Table references are incorporated by reference in the description.
Table i: Promoters active at different stages of spermatogenesis.
Promoter Gene Minimal defined Specification of expression References EF-lalpha [-1200; -lJ Spermatogonies (Furuchi, 1996) LTR Spermatogonies (Dupressoir, 1996) FRMlf [-2800; Spermatogonia lJ

(Hergersberg, Pdha-2 (_lg ~~ _lJ Early Spermatocytes 1995) Hsp70-2 (_640 ~ + 287JLeptotnes spermatides (Iannello, y round 1997}

(Dix, 1996) Sycpl (-54, + SJ Leptotne / zygotne pachytne) present invention CamII (-294; + 68J Spernnatocytes (Ikeshima, . 1994) -1000 ~ 1 SPe ~ primary atocytes ['J (Bartell 1996) H 1 t and spermatids, (34-Gtase (_543; + 253J

Pachytne spermatocytes (Shaper 1994) and round sperma, Zfyl ~ (-4300; -1]

Pachyt sperm (Zambrowicz , and sperrnaaides round 1994) Tcp-lUbt (-973; -lJ Pachytne spermatocytes elongated spermatids (E ~ lonu, 1993) Pgk-2 (-515; -1] Pachytne spermatocytes elongated sperrnatids (Robinson 1989)

8 Hst70 [-800; -1] Pachytous spermatocytes and post-miotic cells (Widlak, 199s) Hox-a.4f (-4000; +1000] Pachytne spermatocytes and post-miotic cells (Berhinger, 1993) Proacrosin (-2300; -1] Round elongated spermatids c-Kit (-6232 ~ + 526] Nayernia, 1994) (16 ~ "'e intron) Round spermatids sperm. {protein truncated) (Albanesi, 1996) ACE [-698; -1] Spermati ~ extensions and sperniatozodes (Langford, 1991) pal [-2400; -1] Spermades alionges and , sperm peschon, 1987 The present invention also relates to transgenic animals susceptible to be obtained by the processes described above =:
s Another object of the present invention is a nucleotide sequence allowing to effectively promote the expression of a gene of interest during the first phase meiotic of the spermatocytes of a mammal characterized in that it comprises at at least one sequence having at least 80% idc; ntity with the sequence SEQ m # 1. Of l0 preferably said nucleotide sequence comprises at least the sequence SEQ
ID # I
or SEQ mn ° 2.
The -54 / + 5 region of the Sycpl promoter (SEQ 1D n ° 3) is the sequence minimum for addressing expression in spermatocytes. The region -260 / + 5 is however 1s required for expression at maximum rate.
Thus we define the minimal Sycpl promoter nucleotide sequence for promote efficient and addressed expression in spermatocytes as comprising at least one sequence having at least 80% identity with the sequence 20 SEQ 1D n ° 1.

WO 00! 12742 PCT / FR99 / 02053

9 SEQ mn ° 1 and SEQ ID n ° 2 are the sequences -260 / + 5 in the mouse and -316 / + 5 in rats respectively. Preferably, this nucleotide sequence is characterized in that it comprises at least the sequence S ~ EQ mn ° 1 or SEQ 1D
# 2. It is obvious s that any sequence equivalent to SEQ ID # 1 and SEQ ID
n ° 2, are targeted by the present invention. By equivalent sequence is meant any sequence of a sufficient mammalian Sycpl promoter to induce effcacement the expression a gene of interest specifically in mammalian spermatocytes, such by 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 coding sequence for a gene of interest. The gene of interest is preferably a gene coding for a recombinase site specific, including Cre. It also relates to a DNA molecule ls 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 2o specifically in spermatocytes, preferably a recombinase specific of site, especially Cre. Recornbinase remains associated with the chromatin of sperm.
This animal is useful for the previously mentioned processes.
The present invention also relates to a method of recombination between chromosomes paternal and maternal 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 several sites) specific) characterized in c; e that the recombinase, expressed during the first phase of meiosis, catalyzes the recombination between sites specific.
In another aspect, the invention relates to an animal capable of being got by this process and a transgenic mufti animal obtained by crossing animals susceptible to be obtained by the methods described will be known. The transgenic animal according to the invention l0 is a mammal, in particular selected by sheep, pigs, cattle, and rodents.
An advantageous aspect of the present invention relates to the use of said animal transgenic for the preparation of substances useful in medicine, in cosmetic, and / or 5 in the food industry, in particular active substances, preferably peptides or polypeptides, antibodies, antigens, lhormones, or growth, and / or for the manufacture of food supplements. For example, we can make produce a protein of interest in mammalian milk under control of a tissue specific promoter; for example WAP rabbit or WAP mouse (request for l0 European patent n ° 92401635.5-2105).
The animal can also be used as an experimental model for identification of genes, promoters, proteins, especially genes involved in diseases genetic, and / or to test the action of active substances, for the preparation organs or cells with better immunocompatibility with 15 humans than organs or cells of wild type or likely to have a weak rejection rate by human immune defenses, and for animal husbandry.
The present invention also relates to all of the elements such as described in the claims, said elements being incorporated into the description by reference.
For the rest of the description, reference is made to the legends of the figures.
presented below below.
LEGENDS OF FIGURES
Figure 1: specific site recombination.
A- Schematic representation of the deletion of specific genomic region through intramolecular recombination between so-called floxed regions located between two sites LoxP.
3o B- Targeted integration of a foreign sequence catalyzed by a recombination intermolecular between two DNAs each carrying a LoxP site.

Figure 2: homiotic meiotic recombination between paternal chromosomes and maternal at a specific LozP site.
Figure 3: activation of the Lo ~ P- ~ 3ge transgene, ~ without promoter after microinjection of construction Pgkl-Loge.
A. The plasmid pGK-LoxP comprises a BamHI-HindIII fragment, of PGK ~ igeobpA
(Friedrich and Soriano, 1991) containing the promoter of the Pgkl gene, inserted between Ies BamHI (B) and HindIII (H) sites of pBSIl2 (Sauer and Henderson, 1990). DNA
plasmid was cleaved with HindIII and PwI (P) before microinjection, which resulted in for consequence of deleting the LoxP site in 5 '. Microinjection has been performed according to standard techniques (Hagan, 1986) in fertilized eggs from crossover between a wild type female and a double transgenic male carrying the transgene Tcp-lObt-cre and LoxP- (3geo.
B, C and D. The nine microinjected cells were kept in culture medium for 24 hours and were stained with X-Gal to detect activity ~ 3-galactosidase. B is the control (no microinjection). None a <: activity ~ endogenous 3-galactosidase is not detected. C and D are two series of eggs in which we have microinjected the construction described in A. The activity ~ i-galactosidase is detected (staining and arrows).
2o Figure 4: insertion specific site of GFP at the Rxra locus.
A. Schematic representation of the genome of the parent male mouse Sycp I -Cre Itxra ° '~~~ ll in somatic cells and gametes.
B. Map of the LoxP-GFP insertion vector. The plasmid was constructed by insertions successive inside the plasmid pBS112 (pBR322 containing a LoxP site, Sauer and Henderson, 1990) from the following fragments - BamHI-Ncoi from pGTl.8 IRES (3; geo (Mountford, 1994) with a part of the En-2 intron of the "engrailed" Drosophila gene, a splice acceptor (Her), and an Ires (Internai Ribozomal entry site).
- HindIII-EcoRI from pEGFP (Ctont ~ ech) containing the coding region for 3o GFP.
- r SacI-Xhoi from pSAj3geo with the polyadenylation signal (pA).

C. Diagrams representing the ~ recornbination carried out by Cre leading to Integration in Rxra ° ~~~ '~ and the modified locus resulting from; recombination site specific.
D. Results of (PCR amplification of (DNA; genomics of 13 babies obtained at go new microinjected cells using primers GFP1 and GFP2, and primers RXRl and int2 (shows the RXR-int junction. The presence or absence of Sycpl-Cre is verified by southern blot.
Figure 5: structure and sequence of the region:>'of the Sycpl gene of the mouse A- Schematic map of the 5 'region of Sycp 1 lo Exons 1 and 2 are numbered, the presence of the first ATG and the site ia initiation transcription (+1) are indicated. The limits of the fragments used for the construction different transgenic lines are represented. The sites of restriction are: B
for BamHI, P for Pst l, S for SacII, and A for ApaI.
B- Comparison of the Sycpl sequence of the mouse (M) and the rat (R).
Putative binding sites for transcription factors are indicated (framed in rectangles). The transcribed sequence (exc> n 1) is underlined.
Figure 6: expression of the various Sycpl / luc constructions in a line somatic cell 2o Each bar represents the average of three experiments, each carried out thrice. The Error bars indicate the standard error to the mean. Values statistics were calculated using Student's test for this figure and the others.
All the results relate to CMV / lacZ internal control. The control vector (pXP 1) contains the gene promoter-free luciferase reporter. The activity of plasmid SV40-luc a been fixed 2s arbitrarily at 100.
Figure 7: Speific expression in the testes of reporter genes in the transgenic lines (-722 / + 102] lacZ, (-260 / + 102] IacZ, [-260 / + i02] luc and [-54 / + 102] luc.
3o Each bar represents the average activity; at least five males transgenic from different lots. Error bars indicate the standard error at the average.
This activity is normalized to the quantity of proteins and to the background noise mice no Wo 00 / I2742 PCT / FR99 / 02053 transgenic belonging to the same lat. The low level of lacZ activity is due to a high endogenous type level (3 galactosida.se in cells testicular. For each of the chimeras, two lines were studied in detail (1 and 2). You represents the testes, Li the liver, Sp 1a spleen, Ki the kidneys, He: the heart and Lu les lungs.
Figure 8: Expression of the luciferase reporter gene in the testes of mouse transgenic [-S4 / + 102] luc and [-260 / + 102] iu <; during their development.
Liver extracts were used as a negative control. Each bar represents the to an average of at least four transgenic males from different lots.
The bars indicate (standard error to mean. P8 to P14 on the axis of abscissa represent the stages of development from the eighth day to the fourteenth day.
Figure 9: Expression of the luc reporter gene in the transgenic mouse j-260 / + S) in different organs.
The specific expression in the testes of the g ~; ne luciferase reporter in mice transgenic [-260 / + 5] luc is shown. Three independent lines have been tested for the expression of the reporter gene. Each b2 ~ r represents the activity average for at minus five transgenic males. Error bars indicate error standard at 2o average among experiences.
The legend of the various organs tested is identical to the legend of the figure 7.
Figure 10: Expression of the luciferase reporter gene in the testes and liver of mice belonging to the transgenic line [-a60 / + 5 ~ during the development.
The horizontally striped bars represent fexpressian in the testes and the diagonal striped bars represent expression in the liver.
P8 to P15 represent the stages of development from the eighth day to fifteenth oven respectively.
Error bars indicate the standard error as an average among experiences.
Figure 11.: Sycpl / lacZ transgenes are not expressed during meiosis in the female.

PCR tests were carried out on the RNA originating from the testes of the liver of the ovary Fetal E17 of transgenic mice [-260 / + 102] lacZ and on ovarian RNA
fetal E16 of transgenic mice [-1848 / + 102) IacZ. RNA was pretreated with DnaseI
and was subject or not to digestion with RnaseA (+ and -) before the reverse transcription. The Sycp l transcripts are detected in adult testis RNA and RNA
from ovaries at the embryonic stage of the sixteenth day (E16) and the seventeenth day (E17), but in the liver RNA. IacZ mRNA is only detected in testicles transgenic adults.
l0 Figure 12: Excision of flogged sequences in the offspring of a male Sycpl-Cre / Rxra ° ~ '~~' NL ~.
Cre sequences identified by PCR amplification with a pair of primers Crel and Cre2 (Rxra wild allele), Rxra ° '~~~' ~ (allele die; Summer), and Rxra ° '~~~' ~ (target allele), with RXRI and RXRII and tk-neo, with tkrieo 1 and tkneo2 (Feil et al. 1996). The column 1 corresponds to the progenitor mouse, and columns 2 to 10 represent babies belonging to the same litter.
Figures x3 and 14: targeted deletion of fl ~ ozed multicopy genes in cells germinal.
2o DNA was prepared from various tissues from double males transgenic progenitor and extract from the tail of their offspring, digested with the enzyme of HindIIi restriction and using pSA plasmid DNA (3geo (column 1 to 7) or by pPGKA (3geobpA (column 8 to 13) as probe, which reveals the floxed sequences and Ies pBR322 sequences of the Cre transgene. Columns 1 to 3 were loaded with DNA
of a double transgenic progenitor Sycpl-Crel [floxed igeo (l represents the liver, 2 kidneys and 3 testes). Columns 4 to 6 correspond to the DNA extracted tails of three babies obtained with a normal mouse. Column 7 corresponds to DNA
of a mouse carrying the trangene Sycpl-Cre (control). Column 8 represents DNA
and the column 9 the testes DNA of a Sucpl / Cre double transgenic progenitor Pgkl 3o floxed. Columns 10 to 13 correspond to the DNA of the tails of four babies obtained. Restriction fragments are indicated by "c" which is fragment of WO OOIiZ742 PCT / FR99 / 02053 Cre transgene; "in" which represents the internal fragments of the transgenes predicted from of their nucleotide sequences (the respective sizes are 3.9 and 2.4 kb for (igeo and 3 for Pgkl); ", j" corresponds to the junction fragments with the sequences cell phones neighboring. The transgene is represented by a single line, the sequence s chromosomal by a double line, and the L, oxP sites by a triangle.
Detailed description.
The original method of the present invention therefore makes it possible to transfer a 1o recombinase in oocytes in order to perform quickly and with a high efficiency all site-specific recombination operations in the genome of mammals, in particular the insertion of a foreign sequence (knock in), the sequence replacement or gene silencing, and recombination meiotic between parental chromosomes (See Figures 1 and 2). This method is based on the 15 surprising demonstration that the recombinase, expressed during meiosis, East transmitted in association with the chromatin of the sperm and is therefore able to catalyze a recombination of a predefined site in the embryo before the first division.
It should be noted that the Cre transgene is present in the father in the state hemizygote. This has as a result that only 50% of the descendants receive it. On the other hand, all 2o receive the enzyme associated with the gamete's chromatin, and the recombination takes place effectively and only in embryos at the i 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 the somatic cells throughout the life of the organism, from development embryonic until his senescence. Thus, the genetic modifications carried out are reproduced at during development in all cells of the body, without chimerism detectable, said changes remaining stable and immediately hereditary. The method presented is applicable to organizations for which the techniques of 3o transgenesis have so far been difficult to develop (absence of lines ES cells).
Transfer of Cre recombinase by sperm chromatin.

In the case of the Tcp-10-bt-Cre transgene first, then in that of the transgene Sycpl-Cre, we have shown that Cre enzyme molecules synthesized during spermatogenesis remain associated with the compacted chromatin of the sperm.
inactive in this context, they can nevertheless perform recombination Between LoxP sites When returning to a decamped 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.
Io The activity of the recombinase was tested in the following e ~ ~ experiments During a first series of experiments, fertilized eggs were obtained from wild type females crossed with hemizygous males for the transgene Tcp-Obt-cre and for multicopy insertion of the floxed coding region of the gene ~ 3geo without Is promoter. A construction with a LoxP site located 3 'from the promoter Pgkl was microinjected (Figure 3A). according to standard techniques (Hogan, 1986). After culture hours, approximately one in four embryos reveals staining positive with X-Gal (Figure 3B, C, and D). The expression of the gene (3l; eo is due to integration effective of promoter microinjected upstream of the cod region <~ nte.
A simpler genetic configuration such as. The structure Rxra ° '~~ L ~ was used in order to highlight the specific site integration of the microinjected DNA.
This second series of experiments demonstrated the presence of a Cre activity the tent 2s in the sperm of the foreign Sycpl-Cre mice, in which excision floxed sequences take place during gametogenesis leaving only one single site LoxP at the time of fertilization. In addition, these experiences have made it possible to to know if recombinase is still active in the male pronucleus and could to permit integration of a floxidized transgene into a predetermined LoxP site. Males who 3o have the Sycp-1-Cre transgene and the Rxroc ° '~ F2 (LNL) locus floxed were obtained (Figure 4A). In the sperm of these males, the tkneo sequences were abundantly excised to generate the recombinant allele Rxra ° '~ tL ~. This allele is transmitted to the whole offspring who only have one LoxP site remaining. Mice double transgenic were crossed with wild type females; the embryos fertilized have been isolated; and a recombinant DNA comprising the sequence coding for the s protein for GFP protein (Green Fluroesce; nt Protein) upstream of a acceptor splicing and an IRES (Internai Ribosomal: Entry Sequence) (Figure 4B) was microinjected. PCR analysis of the genomic structure, presented in figure 4D, watch that the coding sequence for GFP has been inserted inside the locus Rxra ° '~ 2tL ~ (figure 4C) for many babies and no animal keeps the LoxP locus intact.
The lo animals of the Rxra + / + type are under-represented.
The Rxra-LoxP-GFP-LoxP locus resulting from the recombination is transmitted and found in the somatic tissues of the descendants of females and males Crè.
The expression of GFP was demonstrated by fluorescence microscopy in the 15 skin and testicles of these mice. Among the multiple applications offered by Ie efficient transfer of a recombinase by chromatin from sperm, mice rapporteurs, each with a genomic locus carrying an insertion with a website LoxP and a coding region for a reporter protein (GFP, luciferase, (3-galactosidase) can be obtained.
The introduction of a transgene in a single microinjection step can be used at identify a promoter, or analyze a mutant promoter in a context genomics known and in all 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 promoter given.
The method for the targeted insertion of a sequence carrying a LoxP site microinjected in the fertilized egg praduit of the crossing of a double transgenic mast Sycpl-Cre 3o LoxP is presented in Figure 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 fexcisian of the floxed sequence (tkneo) in the sperm of males;
3) a male is crossed with a wild type mouse for the Rxra locus and lo Cre; the enzyme Cre remains associated with the LoxP site of the mutated Rxra allele in the chromatin (50% of embryos); independently 50% of 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 embryos, Cre + or Crs, having received the gene comprising a site LoxP, inserted the region encoding the GFP protein therein thanks to the action of Ia recombinase;
7) expression: the LoxP site had been inserted into an intron of the Rxra 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 (Intemal Ribosome Entry Site) allows the translation of TARN whose 3 'end is defined by the polyadenylavtion signal ("polyA") added downstream of the coding region. The green fluorescence characteristic of the expression of 34 new reporter is highlighted in bodies known for express the Rxra receptor.

In the experiments carried out during the fulfillment of this invention, the recombination efficiency was between 50 and 84% {recornbinants reported to number of mice born after microinjection).
It goes without saying that the process of the present invention, particularly illustrated below 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 expression of a site-specific reeombinase, or site-specific recombinase lo particular.
The range of applications made possible by the transfer of a recombinase by the chromatin of the spermatozoa is notably illustrated by the different case of following figures 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 illegitimate transgenesis and recombination, ie by homologous recombination.
The insert 2o upstream of genomic fragments cloned into a vector comprising also a LoxP site allows, from the first generation of mice, to identify animals so the fragment which express the GFP in the tissue of interest. Several "mice rapporteurs "
can be used in parallel, with genes. different rapporteurs {in particular (3 galactosidase or Iuciferase), at different Chromosomal Locations. We can also insert a gene of interest coding for a protein of interest expressed through example in mammalian milk.
Analysis of a promoter's expression pattern.
3o The insertion of a reporter downstream of a promoter (GFP downstream of Rxra) allows to precisely establish the expression pattern of the intact promoter in his natural genomic localization.

Expression of heterologous genes ("knock in").
According to the expression pattern after (insertion of a reporter gene, we can 5 achieve expression of any biologically active gene under control transcriptional of the promoter of interest. For example, we can insert a gene encoding a toxin, especially diphtheria toxin, under the control of a tissue promoter specific; and thus causing the destruction of possibly given cells at a stage given some development.
Mutant analysis.
The insertion of cassettes comprising different mutants of the same sequence allows to compare their properties in a constant chromosomal context, and in constant or variable physiological conditions.
The Scyp uromotor, 1 can be used in the process according to the invention.
The Sycp 1 gene is expressed only during the prophase of the first 2nd meiotic division in germ cells: males and females (Meuwissen et al, 1992; Heyting et al. 1989; Offenberg et al. 199: 1; Dietrich et al. 1992;
Moens et al.
1992; Dobson et al. 1994 and Dietrich et al. 198.3). The nucleotide sequence of the 5 'region of the mouse Sycp 1 gene has been described by Sage et al. 1997 (Figure 5A). The upstream region of the gene does not contain TA'.CA box, but the site of transcription includes (CCA * 1GTCC initiator element (Sm ~ ale et al. 1989).
genomic library with a Pstl fragment of rat cDNA (692 bp) which covers the 585 bp from the 5 'end of the coding region of Sycpl and a piece of the sequence untranslated leader (Meuwissen et al. 1992; Sage et al. 1995), led to the identification of five genomic clones, one of which includes the 5 'region 3o upstream of the transcription initiation site.

The BamHl fragment extending farthest from the 5 ′ side, which hybridizes with the probe CDNA 692 bp, has been subcloned and its nucleotide sequence has been established. The sequence nucleotide in the region analyzed, extending from positions -324 to +268 show a 80% global identity 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.
Transgenic analysis and several series of deletions at (inside the promoter lo Sycpl made it possible to define two different functional regions. A
strong enhancer is present between positions -260 and -54. A 59 bp proximal element long (-54 to +5), is sufficient to direct (expression of the heterologous reporter enes (lacZ and Luke) in the appropriate male germ cells (Figure 5).
1s Since fenhancer present in the Sycpl promoter is active in bloodlines cell 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 the expression of Luciferase in spermatocytes primary. The sequence comparisons between the four early meiotic grenaes of the mouse 20 (Sycp 1, Hsp70-2, Pdha-2, and Xmr), revealed no conservation of a Liaison site for transcription factors. There is, however, a high degree of similarity (80%) between the Sycp 1 promoter sequences in mice and rats. For example, an E-box and a potential site for Myb-like transcription factors are kept.
25 In the ovaries, where meiosis is initiated during the early stages of development, no expression of reporter genes is observed in Les oocytes. So the Sycpl promoter is very advantageous as a tool to direct the expression of the Cre recombinase, because its proi: it of activity allows the genetic stability organisms and their descendants.
3o Cre-induced recombination at the site. LoxP has been programmed to have location during meiosis in male germ cells. The Cre gene has been expressed as transcriptional control of a promoter region of the Sycpl gene, which is active at WO 00/12742 PCT / F1t99 / 02053 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 who has been used in the construction Sycpl-Cre led to the expression of transgene exclusively in the testicles.
As a result, floxed sites have never been altered in the descendants of females. The floxed sequences were maintained stably in the fabrics somatic (except in the testicles. No expression was detected during stadiums embryonic posterior to (implantation and dan, adult tissues even in using Ia very sensitive RT-PCR technique (Meuwissen et al. 1992).
to Cre-induced recombination in the testes represents a process advantageous compared to that described in previous studies on the activity of the recombinase in other mouse tissues (Akagi et al. 1997; Gu et al. i 994;
Wagner et al. 1997). Indeed, when we test 1; ~ descendants obtained by the process of the present invention, it can be seen that all the floxed alleles transmitted by males 1s underwent recombination. Such a frequency of recombination is particularly suitable for the Cre / lox recombination system.
Cre recombinase can be expressed at the early stage of spermatogenesis.
2e We isolated the promoter of the Sycpl gene (Synaptonemal Complex Protein I), active exclusively during the early stages of meiosis and obtained the expression targeted during zygotene to pachytene stages of reporter genes in mice transgenic. This promoter was used, as well as; that of a promoter meiotic more late (Tcp-10-bt, Ewunolu et al. 1993) to express the CRE enzyme at during 25 spermatogenesis in transgenic mice. In the case of Sycp 1, we finds in males who also carry a floxed transgene, the excision of these sequences during spermatogenesis. This excision does not take place in carrier males of TcplO-bt-Cre transgene. The recombinase is well synthesized, but at a period when begin restructuring and compacting chromatin; what is very 3o probably the cause of the absence of recombination before the formation of the sperm.

Recombination of LoaP sites during your meiosis.
We have shown that (expression from the Sycpl-Cre transgene during the stage meiosis first division pachytene allows recombination between Site (s LoxP at non-identical positions on chromosomes of paternal origin and kindergarten, as shown in Figure 2. Molecular markers available on the paternal and maternal chromosomes show an exchange between chromatids paired with LoxP sites. The frequency of the event recombination in the offspring of Sycpl-Cre males carrying the two floxed chromosomes is of around 30%. From this example, any construction of chromosomes recombined is possible from LoxP sites introduced by conventional methods of homologous recombination in the ES cell of sites inserted by transgenesis and illegitimate recombination, or of sites introduced by addition methods targeted.
Activity of the Sycpl promoter in various cell lines.
We built a series of recombinant DNAs with different parts of the 5 ′ sequence upstream of the transcribed region of Sycp1 (FIG. 5a) linked either to uncomfortable coding for Ia ~ i-galactosidase or for Ia iuciferase. We first performed a 2o search for cell lines which express the Sycpl gene in order to obtain a base for the identification of genetic elements important for the expression pendani meiosis.
We then used a series of cell lines of non-germinal origin for measure the expression of reporter genes after DNA transfection recombinants, in the hope that at least low activity could reveal the presence of promoter.
Unexpectedly, all of these cell lines showed a high level Iuciferase activity after transfection of the various recombinant DNAs (figure 6). These results demonstrate that even the smallest Sycp I fragment extending only up to nucleotide -54 allows the expression of the. luciferase (at one level weak but detectable).

WO 00/12? 42 PCTIFR99102053 A separate activity from the promoter, although not related to an activity specific to meiosis, is therefore controlled by the sequences of the 5 'region next to Sycp I gene. We can therefore conclude that either silencer elements not understood in the longest fragment tested (1950 bp) prevents: the expression of Sycpl in cells somatic in vivo, ie germline specificity; depends on a system of lost signal in somatic cells in culture.
Specific expression in the testes of transgenic sais to Four different fragments of the Sycp 1 promoter were tested for their ability to direct reporter gene expression in mice transgenic ([-1848 / + 102], (-722 / + 102], [-260 / + 102] and [-54 / + 1t72]).
The three longest promoter fragments were tested on the basis of expression of the lacZ gene. Knowing that the low level of expression by the fragment [
1s 54 / + 102] and that the background noise of the activity (3-galactosidase in the extracts from testis is relatively high (Shaper et al. 1594) luciferase has been used in this case as rapporteur. By comparison, the expression of luciferase was tested in parallel for the fragment [260 / + 102]. The results, presented in Table 2 below, demonstrate that in each case at least two family gods express the transgene 20 exclusively in the testicles. This has been observed even for mice who wears it shorter Sycpl sequence [-54 / + 102].

.
, .

v v x U ~

~ '~ O

v ~~ _ .d ~ _ _ _ _ a ~

~~ v at w H ~
0 ~, at. v ~
, .

..

v z w ~ ~

vs. ~ ~
~

o ~ c N

z. ~

w W ~ rn _ O b ~ ' b p, ~ U
s ~ iwv C ~. M ~ - ~ r., M ~ N
~ ti '' O ~ - ~ ~
~

z ~. ~ N ll) CV NNM, O, .w z ~ ' w, ~

~

z '' ~ N ~

c Cr v0 V ~ t O
O fW

.. cM ~ 00 MM ~ '~ N
at .

O

w z C7 v ~ ~, v NN

~ ~ S ~ '~

~ N _ ~ U
~

OOO CV

s .. c ~ "1 -E +

00 ww ~ ... "- + - w,, U
NOO ~ O

~ 00 N ~ l ~~ Jd 'O

~ read-N CV tn N

.

WO 00/12742 PC'f / FR99 / 02053 However, the level of expression is higher. low in families [-54 / + 102], this which implies that the sequence extending to nucleotide -54 exerts a effect in vivo enhancer, as is the case in cell lines somatic s transfected.
This quantitative effect was evaluated by comparing the enzymatic activities in the testicles of mice carrying the luciff reporter; shaved controlled by either the promoter [-541 + 102], or by the promoter [-260 / + 102] of S ~ ycpl. The latter showed way io constant a level of activity 10 to 100 times higher (Figure 7).
Expression during meiosis in adult testes.
The specific expression of ~ i-galactosidase in the testes has been confirmed 15 by X-gal staining of several organs. To identify the types cell phones expressing the transgenes, the activity (3-galactosidase in located in testicle sections.
The positive X-gal cells of animals [-260 / + 102] lacZ correspond to the 2o second layer of germ cells located at the periphery of the tubules.
An over-hematoxylin staining confirmed that lacZ positive cells make part of spermatocytes in the ITi-IV pachytene stage of the seminiferous epithelium cycle (Clermont et al. 1972). At the early zygotene and pachytene stage, more low expression (3-galactosidase could be detected in some sections. None coloring 25 cells were not observed in the post-media compartment of tubules nor for early and diplotene zygatin 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 induction 30 or the suppression of the expression of Sycp1 are localized among the fragments of preamotor analyzed.

In order to determine whether the part of the Sycpl promoter (+1 to +102) present in these fragments play a role in establishing the expression profile of Genoa rapporteurs, two additional transgenic families were generated by s microinjection of recombinant DNA deleted from these fragments, but retaining the initiator of the Sycpl transcription with the upstream sequences up 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 transfected cells (Figure 6) and in animals transgenic (Figures 9 and 10, compared to Figure S).
Regulation during development in prepubertal mice.
The expression of reporter genes in immature animals was measured to demonstrate that the expression of an E; st transgene is identical to that of the gene Sycp 1 endogenous. The first wave of meiosis in the juvenile testes is synchronous with the appearance of the first meiotic cells, leptotene spermatocytes, around 10 days after birth.
The first pachytenic cells appeared on the 14th day and the raw haploid cells after three weeks (Bellvé e, t al. 1977). We have analyzed the transgenic families [-260 / + I02] lacZ, [-260 / + 102] luc, [-260 / + S] luc, and [-54 / + 102) luc.
In situ analysis (Figure 8) detected the expression of ~ 3-galactosidase 2s in primary spermatocytes for mice [-260 / + i02] lacZ in the 13th and lTh day after birth, but not on the 8th "day. On the 10th" day, the expression was detected only on certain mice, and in cases where the result is positive, she was observed only in some tubules. These results have been confirmed by measures performed on transgenic mice [-260 / + 102] luc. No expression of luciferase 3o was not detected before 10 '"' th day after birth.

In the mice [-54 / + 102] luc and [-260 / + Sjjluc, the time profile of the activity of the luciferase is the same, is identical to that of the endogenous gene (Sage and al. 1997).
All of these results led to the conclusion 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 lo (ovary.
In mammals, female gametogenesis takes place during development embryonic and is arrested before birth at the dictyotene stage. When prophase of the first meiotic division, the synaptonérnal complex is morphologically is 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). We .
observe this phenomenon in the fetal ovary during SI ~ '"e and I7è"' ~ days embryonic (E15 and E17). Immunolocation experiments performed on cryosections with a antibody to the rat SCP1 protein (IVieuwissen et al. 1992;
Dietrich et al.
20 1992) detected the SCPI protein (encoded by Sycpl) in ovaries of the E16 and E17 mice. We then analyzed the expression in the ovaries of Genoa rapporteurs expressed by various transgenic families during meiosis male. Nor the in situ detection of ~ -galactosidase; nor the I; a luciferase plus test sensitive only allowed detect expression of the transgene.
25 RT-PCR experiments carried out with the RNA of the E16 ovaries of [-1848 / + 102] lacZ and E17 ovaries of [26I / + 102] IacZ transgenic mice have not allowed to detect the transcripts merged between Sycpl's first exon and the sequence lacZ; while the expression of the endogenous gene has been detected in the same samples (Figure 11).

WO 0 (t / I2742 PCT / FR99 / 02053 One possible explanation for the absence ~ of the expression is (use of a alternative Sycpl promoter in the female germ line, as was shown for the case of the DNA methyltransferase gene (~ viertineit et al. 1998).
To confirm this hypothesis, an used the 5 'RACE technique to determine the initiation site of Sycpl transcription in cells meiotics from the ovaries E16. Twelve clones containing the longest fragments cDNA
among 48 clones analyzed, were sequenced. These sequences indicate clearly that the transcription of cell transcription initiation site germinal 1o female is identical to that previously defined in cells germinal males. he appears as well as the signals which assure the temporal expression and spatial of the gene Sycpl during male meiosis are not sui ~ 'rsants for its expression in the female.
Somatic maintenance of fla ~ xed sequences in male tissues t5 transgenic Sycp1-Cre / LoxP.
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; studies. The results obtained have 20 demonstrated that the stably maintained transgene was not transcribed that in testicles.
Double transgenic lines, which. maintain Sycp1-Cre with a floxed transgene were obtained by crossing a Sycpl-Cre mouse with a mouse whose 25 its genome carries a floxed transgene. Techniques for integrating a floxed transgene in the genome of a mammal are notably described in Kuhn and Schwenk, 1997, Galli-Taliadoros et al. 1995; and Feil et al. 1996. In this case, two target sites have created by microinjection inside the new fertilized cloned DNA
: a tandem repetition of a sequence (3geo flanked; e from LoxP site (Friedrich and Soriano, 30 1991) and a tandem insertion with a low number of copies (less than two copies) of a active promoter (LoxP-Pgkl). A third target site corresponds to the allele Itxrcc ° ~ 2 ~~ L ~

(Feil et al. 1996), into which a LoxP-tk-neo-LoxP cassette has been inserted through homologous recombination in the 8th intron of Rxra. In males carrying of LoxP-J3geo and LoxP-Pkgl transgenes, "Southern blot" hybridization indicates a stability total of these loti in the somatic organs.

A more detailed analysis carried out on Sycpl animals-Rxra ° '~ Zt ~' L ~ a shown that part of the males (2 out of 8 tested) maintain in their DNA
somatic both recombinant and intact floxidized sequences: This observation is agree with the results of the studies on the Sycpl promoter mentioned above.
So the 1o structure of floxed transgenes established at birth remains stable in all somatic organs, even with regard to senescent males. Animals chimeras have been ruled out and further studies have been conducted with males in which the sensitive PCR test did not detect a recombination somatic.
The modified floxed sequences were only detected in each case in the ls testes and in sperm DNA.
Deletion of floxed lochs during yar transmission in Sycpl-Cre / LoxP males.
In the offspring of crosses between double transgenic animals 2o with non-transgenic partners, excision of sequences interveners have always observed when the floxed locus was transmitted by males Sycpl-Cre / LoxP, while the same lots were completely stable when they been transmitted maternally. These results are shown in Figure 12 for the offspring of a wild type female with heterozygous male for floxed allele Rxra ° '°' ~~~~ '~ and 25 hemizygote paur ie transgene Sycpl-Cre. PCR amplification of DNA
extract from the father's tail (column 1) did not detect the presence of floxed sequences (fragment called "tkneo"). None of the nine babies (column; s 2 to IO) have this fragment. This result actually corresponds to two separate situations 30 - Two babies (columns 4 and 10) received the all ~; the wild Rxroc from the father (identified by the characteristic amplification of the product ("Rxra").

- The other babies inherited the Rxra ° allele '''~~ LNL ~ for which tk- sequences neo were excised.
Amplification with the two primers RXI ~ I and RXR2 generated a wider characteristic fragment of the recombined locus ("Rxra ° '°' F2 ~ L ~.
Excision has been noted regardless of the presence (columns 2, 6, .B, 9) or the absence (columns 3, 4, 5, 7,

10) of the Sycpl-Cre transgene.
Similar results were obtained for the multimeric transgene (3-geo-LoxP and the Pgkl-LoxP transgene (Figure 13). Excision in this case is highlighted by Ia lo disappearance of the bands which correspond on the "~ Southern Words" to internal fragments predicted from the transgene sequence ("in"), while the fragments junctions with the endogenous sequences ("j") were, in all cases, maintained.
At the male parents, the floxed sequences were not modified (columns 1, 2, 8, figure i4), with the exception of DNA from the testes (columns 3 and 9).
is Implementation of the method according to the invention with the promoter Tcn-IObt:
Within the framework of the inventian, the promoter of one of the genes of the Tcp- locus l0bt was also used to direct the expression of Cre recombinase in the 2o male germ cells. Those skilled in the art have access to all information necessary in Ewunolu et al., Development, 1993 117: 89-95 on the characterization of Tcp-lObt promoter in transgenic mice. Analysis of the expression of Genoa rapporteurs (for example the gene coding for (3-galactosidase) made it possible to conclude that this promoter makes it possible to mark the population of germ cells by phase 25 late meiosis (late pachytene haploid stage).
We carried out a targeted deletion of gi ~ nes floxed by the transcene Tcp-lObt-Cre. For this purpose, two independent lines carrying the transcene Tcp-lObt-Cre have been established. We first examined the activity of the en: ayme Cre in three types different from 3o "floxed" transgenic mice. Whatever the constructions, a gene without promoter (~ -geo), a promoter not active in the testis (pgk-I) in copies multiple or the tk-neo cassette in single copy introduced by homologous recombination in the Rxroc locus, these remain completely intact in the cells germinal male (including in sperm purified from epidydime).
On the other hand, the majority of the descendants obtained by these males excised the fragment s fioxé. Analysis of recombination in purified speirnatozoids and the descendants of these males showed the presence of the inactive Cre enzyme in these cells germinal by the fact that the Cre recombinase regains its activity following the decompaction of nucleus of sperm in the fertilized egg of the mouse or in a test tube by of biochemical processes.
1o This observation of the maintenance of the Cre protein associated with chromatin compacted from the sperm, from its transfer to the zygote, where it is active in the male pronucleus after fertilization, paved the way for the operation of the insertion of a foreign sequence at a genomic LoxP site; by simple microinjection into the egg fertilized. The relatively simple operation consists in establishing by crossing a male 15 carrying two transgenes. One is Tcp-lObt-Cre, le; second is minimum a LoxP site into a chromosomal locus (if the locus contains two LoxP sequences, the floxed region will be eliminated during the decompaction, bringing back to the case of a single site).
The Cre enzyme will only be synthesized in the late stage of nneiosis, but will be present in all haploid cells, the Cre gene itself being inherited by only 50% of gametes.
2o The presence in the nucleus of the spermatozoon, and clones in the pronucleus of the embryo in stage 1 of a chromosomal LoxP site and of the active recombinase, allows targeted integration of exogenous DNA carrying a LoxP site introduced by microinjection. The eWciency of this integration was measured with two combinations different insert and recipient locus: integration of a promoter (pgkl) upstream 25 of an IacZ coding sequence of the c-myc promoter upstream of the same sequences.
In both cases, the e ~ ciency of integration into the target site was equal or greater than SO
births.
Example 1: Cell culture and transfection experiments.
The BALB / 3T3 cell line (clone A31 American Type Culture Collection CCL-163) and was transfected with 2 µg of the chimeric promoter with 0.1 p, g of CMV-lacZ Control construction, using the co-precipitation method at calcium phosphate {Sambrook et al. 1989). 2: x 104 cells were inoculated by 35 mm box. The enzymatic tests were carried out two days after the transfection.
Example 2: Microinjection and analysis of transgenic mice.
Transgenic mice were generated by microinjection of DNA
inside the new fertilized according to commonly used techniques used by those skilled in the art (Hogan et al. 1986). The animal check foreigners a been performed by Southern and / or Dot block DNA endpoint analysis (Sambrook and have. 1989). All transgenic families have been generated and maintained in one bitter genetic background C57BL / 6xDBAl2 F1.
Example 3: Isolation of the Genomic Clones of Rats The genomic clones of rats were; isolated by bank screening genomics (Sambrook et al. 1989) in a lambda DASH vector (stratagene) with a rat cDNA primer (Meuwissen et al. 1992).
2o Example 4: Analysis of the sequence DNA sequencing was performed using the Sequenase TI kit (Amersham). The Genetic Computer Group's FAST, A computer program has been used for sequence comparisons in databases. The numbers of access to the Genbanl {and EMBL databases for the sequences of promoter in rats and mice are respectively: nt AF020295 and AF020296.
EXAMPLE 5 Plasmid Construction All fragments of the Sycpl promoter come from a genomic clone containing 12Kb of sequences upstream of the transcription initiation site from Sycp l {figure 5) {Sage et al. 1997). At their 3 'end, all the fragments tested from the promoter (with the exception of a fragment, see below) extend to the SacII site which is located in the first exon at position +102 (Sage et al. 1 (95). Cutting the clone genomics with BamHI and SacII led to a fragment of ~ e 2Kb which was then treated with the Klenow fragment of (DNA polymerase (Biolabs) and inserted at the EcoRV site in the vector pBluscript KS (-) (stratagem). A 1950 bp EcoRI-Xhol fragment a then been subcloned into the pnass plasma [3 (clontech, accession number U02433) wearing the lacZ reporter gene ([-1848 / + 102] IacZ pla.smide). ~ n to get chimeras comprising shorter genomic sequences, PCR amplifications were summer made using internal oligonucleotides of 18 bp 5 'primer in conjunction lo with (primer T3 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 plasmi ~ .de pnass ~ 3. The same fragments of promoter were then transferred to the luciferase reporter gene in the plasmid pXP 1 {Lee et al. 1994} (these plasmids have the same names as is previously with "luc" instead of "IacZ". An additional plasmid has summer constructed with the 5 'sequences upstream between positions -260 and +5. A
fragment PCR was amplified with the same 5 'primer used for the chimera [-260 / + 102) lacZ, the other primer covers the Sycpl transcription initiation site {position + 5 / -15). This 275 bp fragment was subcloned into pXPl to form the plasmid [-260 / + 5] Iuc.
EXAMPLE 6 Isolation of the RNA and Analysis Total RNA was prepared according to the isothiocyanate method (Chomezynski et al.
1987). Control RNA samples and Myb '~~ A mice were used (Toscani et al. 1997}. Fetal ovaries from a pregnant woman have been gathered, and to ensure that the RNA preparations are comparable, the same amount of testis and liver was used. Total RNA was treated with DNaseI
(Boehringer), then extracted with phenol and precipitated with ethanol. 2 p, g of RNA was reverse transcribed and amplified using Boringer's Titan kit. lin regarding reactions from control, the RNA was pretreated with RnaseA. The primer sequences used to detect Sycpl transcripts correspond to positions I15-135 and 495-475 in cDNA {Sage et al. 1995}, leading to a product 380 bp long. The WO 00/12742 PCT / F'R99 / 02053 oligonucleotides used to detect the fusion transcripts Sycpl and lacZ
is at position SO-68 in cDNA of Sycpl and at position 444-425 in the pnac plasmid reporter IacZ (3. PCR cycle conditions were 30 seconds at 94 ° C, one minute at SS ° C and 30 seconds at 72 ° C
for 32 cycles. The products of 5 this reaction was hybridized with an internal probe (position 245-26S
in the cDNA
Sycpl and 358-378 in the plasmid pnassj3. Sycpl-lacZ cDNA fi ~ agments have been subcloned in pBluescript and sequenced to confirm correct splicing intron SV40.
The determination of the S 'end of the Sycpl gene in oocytes was performed in lo using the B 'oehringer S' RACE kit following the procedure previously described in Sage et al. 1997.
EXAMPLE 7 Test of the Activity of the Reported Genes: Urs I5 The ~ i galactosidase activity was tested on tissue extracts in using the Galacton substrate as described in Shaper et al. 1994. The determination in situ was performed using an alternative procedure to cryosection, which has allowed a better conservation of the structure of the seminiferous epithelium. Albuginea has been removed and the tissue was carefully stretched with the help of forceps in order to facilitate the 2o penetration of X-gal. Fixation in 2% 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 postexed for 24 hours in 10% of paraformaldehyde and (inclusion was carried out in the "Leica Historesin" in following the manufacturer's instructions (Leica Instruments). 5 p, m sections thick were 25 over-stained with Harris hematoxylin. The luciferase activity was measured using "Luciferase Asay System" (Promega) according to the manufacturer's instructions.
EXAMPLE 8 ImmunolocaIization of the F ~ CPl Protein of the Mouse 3o The cryosection of 8 pm thick of embryonic testes aged 15 or 16 days (E15 or E16) were incubated with directed polyclan antibodies against the rat protein as previously described in Meuwissen et al.

WO OO / I2 ~ 42 PCT / FR99 / 02053 EXAMPLES 9 Construction of the Plasmids and Production of the Mice transgenic.
The plasmid Sycp 1-Cre was constructed; in two steps. A fragment of the promoter [-722 / + 102) of the extending Sycp 1 gene; from -722 to + I02 relatively to the site transcription initiation (Sage et al. 1997, see above) was transferred to the plasmid pBluescript KS (Stratagene) between the EcoRI and Xhol sites. The sequence coding for the Cre recombinase (Genebank Accession number X03453) and a signal polyadenylation was then inserted into the first plasmid digested with the 1o restriction enzymes SaII and KpnI. In the plasmid p ~ igeo-LoxP
(Friedrich and Soriano 1991), the ~ igeo fusion gene was integrated between two LoxP site sites. This plasmid a was constructed by inserting a HindIII-13amHI fragment corresponding to the sequence coding from plasmid p (3geo inside the HindiI-BamHI sites of a plasmid pBS 112 containing two LoxP sites (lFriedrich 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 in inserting the Pgkl promoter inside the HindIII-BamHI sites of pBS112.
EXAMPLES 10 The Transgenic Mice Transgenic mice were obtained ~, es by microinjection of chimeras linearized by PwI inside the new fertilized using techniques well known to a person skilled in the art (Hogan et al. 1986). DNA was purified on columns Qiagen in respecting the manufacturer's instructions, then linearized, and suspended from a concentration 5 ng / ~ .l in sterile water before microinjection. Verification of animals transgenic was performed by Southern and / or dot blot analysis of DNA
extract from the tail of mice. All the transgenic families were generated and kept in a genetic background C57BL / 6 x DBA / 2 FI. The mutant I ~ croc ° ~ 2tLNL ~ has a LoxP-tk-neo-LoxP cassette, inserted by homologous recombination inside the kind 3o intron of the Rxroc gene {Feil et al. 1996).

WO 00! 82742 PCT / FR99102053 Example 11: Expression of Cre transgenes The level of Cre mRNA was estimated by reverse transcriptase analysis {RT-PCR). Isolation of RNA from the kidney liver brain and testicles was performed using phenol aci ~ .dique extraction. The preparations total RNA
were treated with DNaseI (purified of all; residual RNase) and cDNA has been synthesized for 30 minutes at 50 ° C using 50 IU of the reverse transcriptase of Murine Maloney Leukemia Virus (Boehringer Mannheim) using 1 p, g of RNA as a template. It was then amplified for 35 cycles of PCR in using the Crel primers (5'-TGA TGG ACA TGT TCA (iGG ATC-3 ') (SEQ ID No. 4) and Cre2 (S'- CAG CCA CCA GCT TGC ATG A-3 ') (SEQ ID n ° S) constituting a fragment of 865 bp Cre cDNA.
Example 12: Genotyping of the Mouse The end of the mouse DNA was prepared as described in Hogan and 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 phase opened complete reading for the Cre protein using the Crel primers and Cre2. All these 2o fragments were purified twice on agarase gel. Probes can also be prepared from the sequences of the "floxed" transgene.
Example 13: Detection of excision induced by Cre 2s The Southern blot analysis was carried out according to well known techniques of those skilled in the art (Sambrook et ai, 1989). DNA was digested overnight with ies restriction enzymes indicated above (ten times in e ~ s: ces). The fragments of DNA have been then subjected to electrophoresis and transferred to nitroceIlulose. The hybridization have been made using the plasmids pSA ~ igeo or pPK: (3geobpa radiolabelled (Soriano and 3o al, 1991). In addition to "floxed transgenes", the sequences of the plasmid pBr322 present in these chimeras allow the detection of transgér ~ e Cre.

Analysis of double transgenic mice carrying; the Cre expression vector and the allele Rxra ° ~~ L ~ was carried out by PCR amplification. as described in Feil et al. 199b.
With regard to the detection of the Rxra ° 'u ~ t ~ L ~ allele, the sequences floxed tlçneo have were amplified using primers S 'and 3', 5'- i ifiT TCT CCG GCC GCT
TGG GT
3 '(SEQ ff ~ n ° 6) and 5'- GGA GGC GAT GC (J CTG CGA AT-3' (SEQ ID
n ° 7), respectively. With regard to the detection of the Cre transgene, the primers Crel and Cre2 have been used. Visualization of excision of the floxed tkneo marker of the allele target Rxra ° ~~ L ~ was used using the primers RXRl (5'- CCA
GGA GCC
TCC TTT CTC CA-3 ') (SEQ ID n ° 8} and RXR2 (; 5'- CCT GCT CTA CCT GGT
GAC
1o TT-3 ') (SEQ ID n ° 9). These primers amplified a 156 bp fragment from the wild-type Rxra allele and a fragment of a 90 bp from the allele recombined Rxra ° '~~~. The amplified products have been analyzed; by electrophoresis on agarose gel at 2%.

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L = sxE DE s ~ QvEN ~ cEs (~:) INFORM ~ rxioNS GE ~ ~ s (i) DEPOSITOR:
{A) NAME: NATIONAL INSTITUTE OF HEALTH AND
MEDICAL RESEARCH (INSERM) (B) STREET: 101, STREET OF TOLBIAC
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 75654 CEDEX 13 (ü) TITLE OF THE INVENTION: METHOD FOR RESHAPING THE GENOME OF AN ANIMAL
BY ZYGOTIC TRANSFER OF A SITE RECOP ~ IBINASE
SPECIFIC
(i ü) NUMBER OF SEQUENCES: 9 (iv) COMPUTER-DETACHABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / NIS-DOS
(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO) (2} INFORMATION FOR SEQ ID NO: 1:
{i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 392 base pairs (B) TYPE: nucleotide fC) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear {ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERISTIC:
NAME / KEY: -260 / + 5 mice {xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
CATCCACATG AAACACTCTA TAACACGCAC CAGAACATAA TAAP ~ TACAGC CTTGAAAAGG 60 CCAGCATCCT CCGACAATTT TGAGCGCAGA CTTGGTAGAG AACC: CGGCAC GTGGAAGGAA 180 WU 00i12 ~ 42 PCT / FR99 / 02053 (2) INFORMATION FOR SEQ ID NO: 2:
(i} CHARACTERISTICS OF THE SEQUENCE:
{A) LENGTH: 392 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERISTIC:
NAME / KEY: -316 / + 5 rat (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
CATTCACACG AAATACTCAA TAACACAAAC CAGAAAGTAA TAPvATCATTR 60 AFIAAAGAAA

GAAGGAAAGA AA.TGAGACTC CTTTARATGG CTATAAGCCA ATGTGATAGT 120 TGGACGTTAA

GGTTGTGAGC GCCTCAGCTC TTTTGTCTGA GCACCAGCCA GGAAAGCTTT 1 $ 0 CCCAGAGTCC

GGGAAGGGAA

ATTTAGCCAG

(2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A} LENGTH: 186 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERTSTIC:
NAME / KEY: -54 / + 5 mice (xi.) DESCRIPTION OF THE SEQUENCE: SEQ ID NO ~: 3:

(2) INFORMATION 80UR LA SEQ ID NO: 4:
(i) CHARACTERiSTICS OF THE SEQUENCE:
(A) LENGTH: 21 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERISTIC:
NAME / KEY: Crel primer (xi) DESCRIPTION OF THE SEQUENCED: SEQ ID N0: 4:

(2) INFORMATION FOR THE SEQ ID N0: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERISTIC:
NAME / KEY: Cre2 primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 5:

(2) INFORMATION FOR I ~ A SEQ ID NO: 6:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERISTIC:
NAME / KEY: 5 'primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID N0: 6:

(2) INFORMATION FOR?, Pr, SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CHARACTERISTIC:
NONf / KEY: 3 'primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:

(2) INFORMATION FOR SEQ ID NO: 8:
(i) CHARACTERISTTICS OF THE SEQUENCE:
(A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ü) TYPE OF MOLECULE: cDNA
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
CCAGGAGCCT CCTTTCTCCA
(ix) CHARACTERISTIC:
NAME / KEY: primer RXR1 (21 INE'ORMATIONS FOR THE SEQ ïD NO ~ 9 ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 20 base pairs (B) TYPE: nucleotide (C) NUMBER OF STRANDS: single (D) CONFIGURATTON: linear (ü) TYPE OF MOLECULE: cDNA
(ix) CARP, CTERISTICS:
NAME / KEY: primer RXR2 (xi) DESCRIPTION OF THE SEQUENCE: SEQ Ib NO: 9:
CCTGCTCTAC CTGGTGACTT

Claims (31)

1. Method of inserting a foreign sequence into the genome of an animal including the following steps:
a) Expression of a site-specific recombinase in cells germline of a male carrying at least one specific site in its genome, said recombinase remaining associated with sperm chromatin.
b) Fertilization of the oocytes and transfer of the recombinase by said sperm.
c) Microinjection of a recombinant DNA possessing the "site" structure specific-foreign sequence" in the male pronucleus after fertilization.
d) Insertion of said sequence foreign to the specific site in the male pronucleus of the embryo at the 1-cell stage, catalyzed by the recombinase released during decondensation of sperm chromatin. 2. Method according to claim 1 characterized in that said sequence foreign comprises a gene of interest, to be expressed under the control of a promoter Most often is "possibly"
specific tissue(s). 3. Method according to claim 2 characterized in that said sequence foreign contains a reporter gene fused to a promoter of interest. 4. Method according to one of claims 1 to 3 characterized in that said sequence foreign also comprises a splice acceptor (SA) element, a sequence IRES
and/or a polyadenylation signal. 5. Method of replacing an X sequence with a Y sequence in the genome of one animal characterized in that it comprises the following stages:
a) Expression of a site-specific recombinase in cells germline of a mast bearing a “site specific-X sequence-specific site” structure in his genome, said recombinase remaining associated with the chromatin of sperm.
b) Excision of the X sequence during spermatogenesis by recombinase.

c) Fertilization of the oocytes and transfer of the recombinase by said spermatozoa.
d) Microinjection of a recombinant DNA possessing the "site" structure specific-Y-site-specific sequence” in the male pronucleus after fertilization.
e) Insertion of the Y sequence at the specific site in the genome of the embryo at the stadium 1 cell, catalyzed by the recombinase released during the decondensation of the sperm chromatin. 6. Method according to claim 5 characterized in that it allows inactivation of a sequence gene 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 a mutant of the LoxP sequence. 8. Process for transferring Cre in its active form into an oocyte, characterized in that that it involves the following steps:
a) Expression of Cre in germ cells. males with in their genome at least one LoxP site, said protein remaining associated with the chromatin of sperm.
b) Fertilization of the oocytes and transfer of the 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 expression effective in mammalian spermatocytes. 10. Process according to claim 9, characterized in that said promoter includes at at least one region of the Sycp1 promoter, EF-1alpha, IAP, FRM1f, Pdha-2, Hsp70-2, CamII, H1t, .beta.4-Gtase, Zfy1f, Tcp-10bt, Pgk-2, Hst70, Hox-a.4f, Proacrosin, c-Kit, ACE or Prm1. 11. Process according to claim 10, characterized in that said promoter includes at at least one sequence having at least 80% identity the region of the Sycp1 promoter of sequence SEQ ID No. 1. 12. Transgenic animal capable of being obtained by the process according to one of the claims 1 to 11. 13. Nucleotide Sequence to Efficiently Promote Expression of one 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. l. 14. Nucleotide sequence according to claim 13 which consists of the SEQ sequence ID #1 or SEQ ID #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 recombinase specific of site during the first meiotic phase of mammalian spermatocytes comprising a sequence having at least 80% identity with the sequence SEQ ID
#1 fused to 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 the claims 16 or 17 characterized in that it is in the form of a plasmid, of a vector virus, a pseudo-vector, or linear or circular naked DNA. 19. Transgenic animal containing in its genome a nucleotide sequence according 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 sperm chromatin. 23. Use of an animal according to claim 22 in the method of claims 1 to 11. 24. Process of recombination between chromosomes. paternal and maternal characterized in that it comprises the following steps: a) Expression of a recombinase specific of site in the germ cells of a male carrying in its genome one or various specific site(s), b) a said male is crossed with a female animal carrying in his genome one or more specific site(s), said recombinase catalyzing the recombination between specific sites during the first phase of meiosis. 25. Animal obtainable by the process according to claim 24. 26. Multi-transgenic animal obtained by crossing animals according to one of claims 12 or 25. 27. Transgenic animal according to one of claims 12, 25 and 26 characterized in that that it is a mammal, in particular selected from sheep, pigs, the cattle, rodents. 28. Use of an animal according to claim 27 for the preparation of substances useful in medicine, in cosmetics, and/or in the food industry, in particular substances active agents, preferably peptides or polypeptides, antibodies, antigens, hormones, or growth factors, and/or for the manufacture of supplements food. 29. Use of an animal according to claim 27 as a model experimental for the identification of genes, of promoter, of proteins, in particular of genes involved in genetic diseases, and/or to test the action of substances active. 30. Use of an animal according to claim 27 for the preparation organs or cells with better immunocompatibility with humans than them organs or cells that are wild type or likely to have a low level of rejection by human immune defenses. 31. Use of an animal according to claim 27 for breeding.