Transgenic mouse for targeted recombination mediated by modified Cre-ER Number:7,112,715 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to a metazoan organism, with the exception of humans, and in particular a mouse, characterized in that at least one cell of this organism comprises at least one fusion protein between a recombinase Cre and a modified ligand binding domain of the nuclear estrogen receptor alpha, allowing the inactive fused recombinase to be induced by synthetic antiestrogens, but not by natural estrogens, and one or more DNA sequences of interest belonging to the genome of said organism into which one or more sites of recognition of said recombinase protein are inserted. The invention also covers the methods using said organism for the screening of medicaments, the mutagenesis and the analysis of the biological function of the DNA sequences) of interest, in particular of gene(s) of interest, such as RXR.sub..alpha..<H recombination, Transgenic, Transgenic, mous, 7112715.html, Patent, pto, 7112715

Title: Transgenic mouse for targeted recombination mediated by modified Cre-ER

Abstract: The present invention relates to a metazoan organism, with the exception of humans, and in particular a mouse, characterized in that at least one cell of this organism comprises at least one fusion protein between a recombinase Cre and a modified ligand binding domain of the nuclear estrogen receptor alpha, allowing the inactive fused recombinase to be induced by synthetic antiestrogens, but not by natural estrogens, and one or more DNA sequences of interest belonging to the genome of said organism into which one or more sites of recognition of said recombinase protein are inserted. The invention also covers the methods using said organism for the screening of medicaments, the mutagenesis and the analysis of the biological function of the DNA sequences) of interest, in particular of gene(s) of interest, such as RXR.sub..alpha..

Patent Number: 7,112,715 Issued on 09/26/2006 to Chambon, et al.

Inventors: Chambon; Pierre (Blaesheim, FR), Metzger; Daniel (Strasbourg, FR)
Assignee: Gie-Cerbm, Centre Europeen de Recherche en Biologie et en Medecine (GIE) (Illkirch, FR)

Appl. No.: 09/853,033

Filed: May 11, 2001

Foreign Application Priority Data


Oct 03, 2000 [FR]			00 12570

Current U.S. Class: 800/3 ; 800/18; 800/21

Current International Class: G01N 33/00 (20060101)

Field of Search: 800/3,18,22 530/350

References Cited [Referenced By]

U.S. Patent Documents


6093873	July 2000	Chambon et al.

Foreign Patent Documents


0 698 392	Feb., 1996	EP
WO 92 06104	Apr., 1992	WO
WO 94 26100	Nov., 1994	WO
WO 95 00555	Jan., 1995	WO
WO 97 10819	Mar., 1997	WO
WO 97 31108	Aug., 1997	WO
WO 99 18222	Apr., 1999	WO
WO 99 25851	May., 1999	WO
WO 00 49147	Aug., 2000	WO

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Primary Examiner: Qian; Celine
Attorney, Agent or Firm: Foley & Lardner LLP

Claims

The invention claimed is:

1. A method for producing spatio-temporally-controlled site-specific somatic recombinations in a mouse, wherein one or more gene or intergenic DNA sequences of interest naturally belonging to the genome of said mouse have been recombined, comprising: a) obtaining a transgenic mouse, wherein said transgenic mouse comprises a transgene encoding: (i) a Cre fusion protein comprising sequentially: (1) a Cre recombinase protein; a hinge region of at least 15 amino acids; a polypeptide comprising the ligand binding domain of the human nuclear estrogen receptor, or of a vertebrate nuclear estrogen receptor, said polypeptide exhibiting at least one mutation relative to the wild-type form of said ligand binding domains; and (2) said Cre fusion protein has a negligible, or even zero recombinase activity in the absence of a synthetic ligand endowed with antiestrogenic activity, the recombinase activity being induced by low dose of the synthetic ligand; (ii) one or more gene or intergenic DNA sequences of interest, naturally belonging to the mouse genome, flanked by one or more recognition sites for a Cre recombinase protein, b) administering to said transgenic mouse a low dose of said synthetic ligand in order to induce Cre-mediated recombination; and c) said gene or intergenic DNA sequences of interest undergo a site specific somatic recombination, as a result of the induction by said synthetic ligand, in at least 90% of the targeted cells of said mouse, whereas said gene or intergenic sequences of interest underwent recombination in less than 5% of the targeted cells of said mouse before step b).

2. The method of claim 1, wherein said one or more sites of recognition specific for said Cre recombinase protein comprise the sequences Lox P.

3. The method of claim 1, wherein said hinge region comprises all or part of the D hinge region of a nuclear estrogen receptor.

4. The method of claim 3, wherein said hinge region comprises amino acids 282 to 301 of the sequence of SEQ ID NO. 2.

5. The method of claim 1, wherein said polypeptide chosen from the ligand-binding domain of the nuclear human estrogen receptors is the ligand-binding domain of the human nuclear estrogen receptor .alpha. and wherein said ligand-binding domain exhibits at least the following mutations: mutation (G400V) glycine to valine at position 400 of the sequence SEQ ID No. 2; and mutation (methionine-leucine) to (alanine-alanine) situated at position 543 544 (M543A/L544A mutation) of the sequence SEQ ID No. 2.

6. The method of claim 1, wherein said transgene is under the control of expression elements ensuring its expression in the targeted cells of said mouse.

7. The method of claim 6, wherein said expression elements are chosen from elements controlling tissue-specific and cell-specific expression or ubiquitous expression.

8. The method of claim 6, wherein said expression elements controlling expression are chosen from elements controlling expression ensuring constitutive expression or elements controlling expression ensuring inducible expression.

9. The method of claim 6, wherein said expression element is chosen from the group composed of the promoter regions of cytokeratin 14 (K 14), of cytokeratin 5 (K 5), and of the adipocyte fatty acid binding protein 2 (aP2).

10. The method of claim 6, wherein said transgene has the sequence SEQ ID NO:5, which encodes the fusion protein Cre-ERT.sup.T2 having the sequence SEQ ID NO:6.

11. The method of claim 1, wherein said DNA sequence of interest comprises the RXR.alpha. gene.

12. The method of claim 1, wherein the genome of said mouse comprises: a transgene encoding the fusion Cre-ERT.sup.T2 having the sequence SEQ ID NO:6, said fusion protein being selectively expressed in adipocytes under the control of the adipocyte fatty acid binding protein 2 (aP2) promoter; and one or more chromosomal DNA sequence of interest in their natural chromatin context and flanked on each sided by one lox site, the two lox sites being oriented as a direct repeat.

13. The method of claim 1, wherein the synthetic ligand endowed with antiestrogenic activity is selected from the group consisting of Tamoxifen, 4-hydroxyTamoxifen, ICI 164 384 and ICI 182 780.

14. The method of claim 13, wherein the synthetic ligand endowed with antiestrogenic activity is Tamoxifen or 4-hydroxyTamoxifen.

Description

The present invention relates to a metazoan organism, with the exception or humans, and in particular a mouse, characterized in that at least one cell of this organism comprises at least one fusion protein between a recombinase Cre and a modified nuclear estrogen receptor allowing it to respond to synthetic antiestrogens but not to natural estrogens, and one or more DNA sequences of interest belonging to the genome of said organism into which one or more sites of recognition of said recombinase protein are inserted. The invention also cover the methods using said organism for the mutagenesis and the analysis of the biological function of the DNA sequence(s) of interest, in particular of the gene(s) of interest, such as RXR.sub..alpha..

The ability to modify the genome of animals, more particularly of mice, by integrating transgenes randomly or at preselected sites, by homologous recombination, in embryonic stem cells (ES cells) has made it possible to greatly improve our understanding of the biological function of mammalian genes under normal and/or pathological conditions (Jaenisch, 1988; Capecchi, 1989). However, these techniques have proved not to be very informative in a large number of cases, in particular because the hereditary mutations thus generated were lethal during development and/or because their effects were pleiotropic.

To remedy these defects, strategies for conditional somatic mutagenesis have been developed, particularly in mice; they make it possible to selectively induce mutations in a given cell type (spatial control) or at a given time (temporal control) during the life of the animal.

A first strategy consists in combining the targeted homologous recombination with the site-specific recombination systems based on the use of recombinases which catalyze the recombination reaction between two short recognition DNA sequences. It has been shown that these site-specific recombination systems, although of microbial origin for the majority, could function in higher eukaryotes, such as plants, insects and mice (Sauer, 1994; Rajewsky et al., 1996; Sauer, 1998). Among the site-specific recombination systems commonly used, there may be mentioned the Cre/Lox (Sauer, 1998) and FLP/FRT (Kilby et al., 1993) systems. The strategy normally used consists in inserting the loxP (or FRT) sites into the chromosomes of ES cells by homologous recombination, or by conventional transgenesis, and then in delivering Cre (or FLP) for the latter to catalyze the recombination reaction. The recombination between the two loxP (or FRT) sites may be obtained in ES cells (Gu et al., 1993) or in fertilized eggs (Araki et al., 1995) by transient expression of Cre or using a Cre transgenic mouse (Lakso et al., 1992; Orban et al., 1992). Such a strategy of somatic mutagenesis allows a spatial control of the recombination, because the expression of the recombinase is controlled by a promoter specific for a given tissue or for a given cell. However, this strategy also has limitations because some somatic alterations can lead to a lethal phenotype at an early stage of development, thus preventing any subsequent biological or physiological study. Also, an insufficiently specific expression of the recombinase can lead to recombination events in a non-desired cell type (Betz et al., 1996) which, if they occur early during embryogenesis, can cause recombination of the DNA in the majority of the adult tissues, and thereby complicate the analysis of the mutant phenotype.

A second strategy has consisted in controlling the expression of recombinases over time so as to allow temporal control of somatic recombination. To do this, the expression of the recombinases is controlled by inducible promoters (Kuhn et al., 1995; Saint-Onge et al, 1996), such as the interferon-inducible promoter, for example. This system also has limitations because it does not make it possible to obtain spatial control of the recombination.

The coupling of the tetracycline-inducible expression system developed by H. Bujard (Gossen et al., 1992; WO 94 04672; EP 804 546) with the site-specific recombinase system has made it possible to develop a system for somatic modification of the genome which is controlled spatiotemporally. Such a system is based on the activation or repression, by tetracycline, of the promoter controlling the expression of the recombinase gene. Such a method, although making it possible to obtain a spatiotemporal control of the somatic recombination, has the disadvantage of being cumbersome to carry out because it requires the creation of a doubly transgenic animal.

It has been possible to envisage a new strategy following the development of chimeric recombinases selectively activated by the natural ligand for the estrogen receptor. Indeed, the observation that the activity of numerous proteins, including at least two enzymes (the tyrosine kinases c-abl and src) is controlled by estrogens, when the latter is linked to the ligand-binding domain (LBD) of the estrogen receptor .alpha. (ER.alpha.) (Jackson et al., 1993; Picard et al., 1994) has made it possible to develop strategies for spatiotemporally controlled site-specific recombination (Logie et al., 1995; Metzger at al., 1995). However, to use such chimeric recombinases to successfully carry out conditional somatic mutagenesis in vertebrates (in particular mice) which produce estrogens, it was necessary to create recombinases which are not activated by the estrogens present in the animal, otherwise the temporal control of the recombination of the target genes would not be obtained. Thus, mutations were introduced into the ER.alpha. LBD, and it has been shown, in cells in culture, that the chimeric recombinase Cre-ER.sup.T no longer responds to the natural estrogens, despite being efficiently activated by antiestrogens such as 4-hydroxytamoxifen (OHT) (Feil et al., 1996).

The feasibility of the site-specific somatic recombination activated by an antiestrogenic ligand has thus been demonstrated for "reporter" DNA sequences, in mice, and in particular in various transgenic mouse lines which express the fusion protein Cre-ER.sup.T activated by Tamoxifen (Tam) (Fail et al., 1996; Brocard et al., 1997; Indra et al., 1999). The feasibility of the site-specific recombination activated by a ligand for a gene present in its natural chromatin environment has been demonstrated in mice by Schwenk et al. (1998). Schwenk et al. have thus carried out the deletion, inducible by injection of Tamoxifen, of a pol.beta. gene in B cell: using a mouse expressing, specifically in the B lymphocytes, a fusion protein between the recombinase Cre and the ligand-binding domain of the mutated human nuclear estrogen receptor. However, the technology developed by Schwenk et al. does not make it possible to obtain a satisfactory efficiency of spatiotemporally controlled site-specific recombination in cells expressing the fusion protein because the efficiency varies between a few percents and 80%, in spite of the use of high doses of OHT (five infections of 8 mg). Moreover, the results presented by Schwenk et al. show that in the absence of synthetic ligand, the activity of the fusion protein used may be induced by natural ligands generating a non negligible residual "background noise" which may be detected by PCR.

Up until now, the possibility of carrying out the ligand-activated, site-specific somatic recombination of chromosomal DNA sequence(s), in their natural chromatin environment, has therefore never been able to be satisfactorily demonstrated in animals, in particular mice, that is to say with a very high efficiency in the presence of synthetic ligand and with a negligible or even zero "background noise" in its absence.

As highlighted by Schwenk et al. in the discussion in their article, there is a real need to develop transgenic animals in whose cells site-specific recombination could be spatiotemporally induced with an efficiency of close to 100% in the presence of synthetic ligand, and which could not occur in any cell in the absence of synthetic ligand and/or in the presence of a natural ligand.

Moreover, a need also exists to develop chimeric recombinases with increased sensitivity to the synthetic ligand, so as to avoid injecting into the animals massive doses of synthetic ligand which can cause in these animals not only unjustified suffering, but can also affect the general metabolism of the animal, which would distort subsequent physiological and behavioral studies.

Unexpectedly, the inventors have solved the problems mentioned earlier, which had not been resolved up until now, by combining the selection of novel mutations in the ligand-binding domain of the human nuclear estrogen receptor, the selection of a suitable hinge region between the two domains of the chimeric recombinase, and the selection of promoters suitable for directing the expression of the chimeric recombinase in a given tissue.

The present invention therefore relates to a metazoan organism, with the exception of humans, characterized in that at least one cell of said organism comprises at least; (i) one fusion protein comprising sequentially: a recombinase protein; a hinge region of at least 15 amino acids; a polypeptide comprising the ligand-binding domain of the human nuclear estrogen receptor, or of a vertebrate nuclear estrogen receptor, and their natural variants or one of their fragments, said polypeptide exhibiting at least one mutation relative to the wild-type form of said ligand-binding domains, or of their natural variants, or of their fragments, and said fusion protein having a negligible, or even zero, recombinase activity in the presence of a natural ligand, such as for example estradiol, and a recombinase activity induced by a small quantity of synthetic ligand endowed with antiestrogenic activity, such as for example Tam and OHT; (ii) one or more gene or intergenic DNA sequences of interest naturally belonging to said genome of said organism into which one or more recognition sites of said recombinase protein are inserted, said DNA sequence (s) of interest being located in one or more of the chromosomes of the genome of said cell.

The expression "metazoan organism" is understood to mean any animal organism, with the exception of humans, consisting of several cells. According to a preferred embodiment, it is a vertebrate such as for example a mammal, a bird, a fish. Preferably, it is a mammal such as for example a bovine, a porcine, a caprine, an ovine, an equine, a rodent. According to a more preferred embodiment, it as a rodent such as mice or rats.

The expression "recombinase protein" is understood to designate recombinases of the family of integrases which catalyze the excision, insertion, inversion or translocation of DNA fragments at the level of specific sites of recognition said recombinases (Sternberg et al., 1986, Sauer, et al., 1990; Barbonis et al., 1993; Kilby et al., 1993; Sauer, 1994; Denisen et al., 1995). These recombinases are active in animal cells (Sauer, 1994).

The recombinase protein of the invention is preferably selected from the group of site-specific recombinases composed of the Cre recombinase of bacteriophage P1, the FLP recombinase of Saccharomyces cerevisiae, the R recombinase of Zygosaccharomyces rouxii pSR1, the A recombinase of Kluyveromyces drosophilarium pKD1, the A recombinase of Kluyveromyces waltii pKW1, the integrase .lamda.Int, the recombinase of the GIN Recombination system of the Mu phage, of the bacterial .beta. recombinase (Diaz et al., 1999) or a variant thereof.

The Cre ("cyclization recombination") recombinase which is a 38 KDa integrase of bacteriophage P1 catalyzes, in the absence of cofactors, recombination between two DNA sequences of 34 basepairs called "loxP site" (Sauer et al., 1990). The position on one or more DNA molecules and the orientation of the loxP sites relative to each other determine the-type of function of the Cre recombinase: excision, insertion, inversion or translocation. Thus, the recombinase activity of Cre is an inversion when two loxP sites are inverted on the same DNA fragment, and an excision when the loxP sites are in the form of a direct repeat on the same DNA fragment. The activity of the recombinase is an insertion when the loxP site is present on a DNA fragment, it being possible for a DNA molecule such as a plasmid containing a loxP site to be inserted at the level of said loxP site. The Cre recombinase can also induce translocation between two chromosomes provided that a loxP site is present on each of them (Babinet, 1995). More generally, the Cre recombinase is therefore capable of catalyzing recombination between one or more different DNA molecules provided that they carry loxP sites.

The FLP recombinase of the FLP/FRT system is a recombinase of 43 KDa from Saccharomyces cerevisiae which is capable of the same type of action as the Cre recombinase on DNA fragments containing FRT recognition sites (Kilby et al., 1993).

Preferably, the recombinase according to the invention is the Ore recombinase of bacteriophage P1 and its natural or synthetic variants, and said sites of recognition specific for said Cre recombinase are preferably chosen from the group composed of the sequences Lox P, Lox 66, Lox 71, Lox 511, Lox 512, Lox 514.

The expression "variant of the recombinase protein"is understood to mean all the wild-type recombinases or fragments thereof which may exist naturally and which correspond in particular to truncations, substitutions, deletions and/or additions of amino acid residues. These recombinases and fragments thereof are preferably derived from the genetic polymorphism in the population. The expression "recombinase fragment" is understood to mean any recombinase portion exhibiting at least one recombinase activity. The expression variant of the recombinase protein is also understood to mean the synthetic variants for which the above modifications are not naturally present, but were introduced artificially, by genetic engineering for example. Thus, the recombinases derived from chimeric fusion constitute synthetic variants according to the invention. Such recombinases have been described for example in Shaikh and Sadowski (2000).

Said hinge region according to the invention comprises the D hinge region of the nuclear estrogen receptor, preferably the human nuclear estrogen receptor .alpha., or one of its fragments.

The D hinge region (region 263 to 301 of the sequence SEQ ID No. 2) is a region situated between the ER C region which contains the DNA-binding domain (region 180 262 of the sequence SEQ ID No. 2) and the ligand-binding domain (region 302 to 552 of the sequence SEQ ID No. 2).

Preferably, this hinge region sequentially comprises at least (i) two amino acids corresponds to the introduction of a restriction site, of a "linker", or of an adapter, which are necessary for the cloning of the fusion gene, and (ii) one fragment of the D hinge region of the human nuclear estrogen receptor .alpha., corresponding to amino acids 282 to 301 of the sequence SEQ ID No. 2. Preferably, said restriction site is an XhoI site and the two corresponding amino acids are leucine and glutamine.

The hinge region according to the invention has a size of at least 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 40, 45, 50, 55, 60, 65, 70, 100, 150, 200, 250, 292 amino acids. According to a preferred embodiment, said hinge region comprises at least 15 amino acids and at most 54 amino acids. More preferably still, the hinge region comprises 23 amino acids. The width of the hinge region influences the regulation of the recombinase activity by the ligand.

The hinge region according to the invention may consist of a peptide which is functionally equivalent to said D hinge region.

The estrogen receptors (ER) are proteins regulating the transcription of genes which mediate the action of estrogens in the target cells. The ERs belong to the superfamily of nuclear receptors which have a common modular structure: (i) a variable N-terminal A/B region containing the constitutive transactivation activity AF-1, (ii) a DNA-binding central domain C (DBD) which is highly conserved between various species and allowing binding of the receptor to its specific DNA response element, (iii) and a ligand-binding domain (LBD), located in the C-terminal region of ER (for review articles and references see Evans, 1988; Beato et al., 1989; Gronemeyer, 1991; Green and Chambon, 1988; Parker, 1993; Simons, 1994).

The nuclear estrogen receptors according to the invention are chosen from the human nuclear estrogen receptors, and from the nuclear estrogen receptors of vertebrates such as for example the various species of primates, bovines, porcines, ovines, caprines, felines, canines, equines, birds, fish, rodents, in particular rats and mice.

According to a preferred embodiment, the source organism for the estrogen receptor according to the invention is characterized in that said ligand-binding domain (LBD) of the nuclear estrogen receptor, or its natural variants, or one of their fragments, is human and is chosen from the LBDs of the human nuclear .alpha. and .beta. estrogen receptors (ER.alpha. and ER.beta.). According to a further preferred mode, this includes the LBD of the human nuclear estrogen receptor .alpha. corresponding to amino acids 302 to 552, or its natural variants, or one of their fragments.

The expression "natural variant" is understood to mean all the LBDs of the nuclear estrogen receptors or their fragments which may exist naturally, in particular in human beings, and corresponding in particular to truncations, substitutions, deletions and/or additions of amino acid residues. These natural variants are derived in general from the genetic polymorphism in the population, and have an activity which is not substantially modified compared with the wild-type receptor.

There are also included in the scope of the invention the polypeptides homologous to the LBDs of the wild-type nuclear estrogen receptors, or to their variants, or to one of their fragments, and which exhibit certain modifications, in particular a deletion, addition, substitution of at least one amino acid, a truncation, an extension and/or a chimeric fusion.

The expression "nuclear receptor fragment" is understood to mean any portion of the nuclear estrogen receptor LBDs exhibiting at least the LBD activity.

Said fusion protein according to the invention is therefore preferably Cre-ER and comprises the Cre recombinase protein to which is fused a portion of the D hinge region and the LBD (amino acids 282 to 595 of the sequence SEQ ID No. 2) of the mutated human nuclear estrogen receptor .alpha. (SEQ ID No. 2). The fusion protein according to the invention comprises at least the portion of the nuclear estrogen receptor having a ligand binding activity.

Said LBD of the nuclear receptor, or one of its fragments, has at least one mutation. This mutation is preferably chosen from the group: mutation (G521R) glycine to arginine at position 521 of the sequence SEQ ID No. 2 or of a natural variant of this sequence; mutation (G400V) glycine to valine at position 400 of the sequence SEQ ID No. 2 or of a natural variant of this sequence; mutation (methionine-leucine) to (alanine-alanine) situated at position 543 544 (M543A/L544A mutation) of the sequence SEQ ID No. 2 or of a natural variant of this sequence.

The term "mutation" is understood to mean any changes occurring in the sequence of the nuclear estrogen receptor, and in particular of the human nuclear estrogen receptor .alpha., other than those present in its natural variants, and/or in its human or vertebrate homologous, and which substantially modify the activity of the recombinase protein fused to said receptor or to said ligand-binding domain, in response to the binding of a synthetic ligand endowed with antiestrogenic activity.

Among the mutations capable of being introduced into the LBD of the nuclear estrogen receptor, there may be mentioned point mutations, deletions, insertions, substitutions However, it is advisable to select only the mutations introduced into the LBD of the nuclear estrogen receptors which allow induction of the activity of the Cre recombinase fused to said receptor by a synthetic ligand in low concentration, while avoiding as far as possible the inducing of a basal activity by the natural ligands for this receptor which are naturally present in the metazoan organism. Likewise, it will be advisable to select the mutations which do not confer activity on the Cre recombinase fused to said receptor in the absence of a ligand.

The G521R mutation constitutes an LBD mutation of the ER according to the invention. This mutation is similar to the G525R mutation introduced into the mouse ER LBD (mER) which reduces the affinity for the natural ligand, estradiol, by about 1000 fold, without adversely affecting the binding of the synthetic ligand, 4-hydroxyTamoxifen (OHT) (Danielan et al., 1993). Thus, the inventors have shown that the recombinase activity of the Cre-ER.sup.T (T=for inducible by Tamaoxifen) fusion protein which carries the G521R mutation, and the amino acid glycine at position 400, called Cre-ER(GR) in the article by Feil et al. 1997, is dependent on the addition of OHT or of Tam to the medium for culturing transfected cells. On the other hand, no recombinase activity is observed in the presence of OHT when the fusion protein carries the G521R mutation and the G400V mutation (mutant called Cre-ER(VR) in Feil et al., (1997)).

The inventors have also created the fusion protein corresponding to the triple mutant G400V/MS43A/L544A called Cre-ER.sup.T2 (Feil et al., 1997). This fusion protein exhibits a recombinase activity in cultured cells which is induced by the antiestrogen Tam or OHT, but not by the natural ligand estradiol; moreover, the maximum activity of Cre-ER.sup.T2 is induced for Tam or OHT doses less than those necessary to activate Cre-ER.sup.T. This increased sensitivity to Tam or OHT of Cre-ER.sup.T2 compared with Cre-ER.sup.T has been verified in transgenic mice selectively expressing the chimeric recombinases in the basal layer of the epidermis, under the control or the cytokeratin 5 promoter (Indra et al., 1999), The Inventors have observed that the translocation of Cre-ER.sup.T2 from the cytoplasm into the nucleus, as well as the excision of the DNA sequences flanked by loxP sites from a "reporter" gene are induced at doses of about ten times less than those necessary for Cre-ER.sup.T.

With the aim of further increasing the sensitivity of the chimeric recombinase Cre-ER.sup.T2 to Tamoxifen, the inventors replaced the valine at position 400 in Cre-ER.sup.T2 with a glycine. This novel fusion protein which corresponds to the double mutant M543A/LS44A called Cre-ER.sup.T3 exhibits increased sensitivity to the synthetic antiestrogenic ligand such as Tam and OHT, without the recombinase activity of this protein being induced by the natural ligand estradiol.

The inventors have thus shown that for 10 times lower injected Tam doses, the recombinase activity in the cells of a transgenic Cre-ER.sup.T3 mouse is greater than that of a Cre-ER.sup.T2 mouse (see Example 5).

According to a preferred embodiment, the fusion protein according to the invention is Cre-ER.sup.T2 whose ER LBD exhibits the mutation G400V/M543A/L544A According to another preferred embodiment, the fusion protein is Cre-ER.sup.T3, whose ER LBD exhibits the mutation M543A/L544A.

One of the objects of the present invention is therefore to provide a Cre-ER fusion protein which exhibits mutations in the human ER.alpha. LED which are preferably chosen from the mutations G521R, G400V, M543A and L544A, whose recombinase activity is not induced by the natural ligands, and is highly induced by a small quantity of synthetic antiestrogenic ligand. Preferably, this fusion protein is Cre-ER.sup.T, Cre-ER.sup.T2, Cre-ER.sup.T3. The present invention also relates to said fusion gene encoding said protein, said vector for expressing said protein, as well as the corresponding host cell, and the corresponding transgenic animal, which expresses said fusion protein in a particular cell type, preferably the epidermis, the liver or the adipose tissue.

The Cre-ER fusion protein of the present invention therefore comprises all or part of a nuclear estrogen receptor and a recombinase protein whose activity is inducible more strongly by the binding of said receptor or of said ligand-binding domain (LBD) of said receptor with a said antiestrogen than with a natural ligand. Said Cre-ER fusion protein makes it possible to carry out a recombination between loxP sites, in a cell of the organism of the invention, following treatment with an antiestrogen. In the absence of treatment, or in the presence of concentrations of ligands such as the natural estrogens of up to 10.sup.-6 M, no excision is observed. This system therefore makes it possible to release the recombinase activity of the chimeric protein at a given and chosen moment. Said Cre-ER fusion protein may be expressed in cells containing loxP sites, without modifying the locus containing the loxP sites. The recombination at the level of the loxP sites takes place only after treatment with an antiestrogen such as Tam or OHT. Furthermore, by expressing said Cre-ER fusion protein in an organism according to the invention, preferably an animal, under the control of a promoter with cellular specificity, it is possible to obtain recombination between loxP sites, specifically in these cells.

The expression "synthetic ligand" is understood to mean any type of compound capable of binding to the nuclear estrogen receptor, and exhibiting agonist and/or antagonist activities, according to the species, the tissue or the cell type. Preferably, and with no limitation being implied, the synthetic ligand according to the invention is endowed with antiestrogenic activity, it is preferably the antiestrogenic therapeutic agent Tamoxifen (Tam), but also its metabolite 4-hydroxyTamoxifen (OHT). The antiestrogens ICI 164 384 and ICI 182 780 are also synthetic ligands according to the invention.

The present invention therefore provides a transgenic metazoan organism and more particularly a transgenic animal, and in particular a transgenic mouse: (i) in which at least one cell contains one or more chromosomal DNA sequences which are present in their natural chromatin context and are flanked (floxed) by loxP sites; (ii) which preferably expresses a chimeric Cre recombinase in a tissue-specific manner in one or more cell types of the organism (iii) whose chimeric Cre recombinase activity is negligible, or even zero, in the presence of estrogen; (iv) whose chimeric recombinase activity is activated by low concentrations of an antiestrogen (from 0.001 to 1 mg of Tamoxifen/mouse/day, for five days); (v) and finally whose Cre recombinase is capable of catalyzing, with an efficiency close to 100%, the site-specific targeted somatic recombination in the nucleus, in a natural chromatin environment of the floxed DNA sequence(s).

The doses of synthetic ligand injected into the metazoan organism according to the invention are low. The term low is understood to mean quantities of less than or equal to 4 mg/adult mouse/day, preferably less than or equal to 2 mg/adult mouse/day, in a preferred manner less than or equal to 1 mg/adult mouse/day. According to an even more preferred mode, this quantity may be less than or equal to 0.5 mg, 0.25 mg, 0.10 mg, 0.075 mg, 0.05 mg, 0.025 mg, 0.001 mg per adult mouse and per day.

It is clearly understood that persons skilled in the art will be able to adjust these quantities, according to the organism, its weight and its age.

The efficiency of the targeted somatic recombination is estimated by techniques known to persons skilled in the art. This efficiency is estimated by the frequency of recombination events catalyzed by said recombinase. These events may be revealed by PCR or Southern Blotting; the recombination frequency being estimated by taking the ratio of the representation of the various alleles in the cells of a tissue. The frequencies of the various alleles may be estimated by assaying the intensity of the corresponding bands on an electrophoresis gel of a product of PCR amplification or of genomic DNA (Southern blotting).

The use of the PCR makes this method of estimation extremely sensitive and makes it possible to detect the presence of cells of the organism whose genome has not undergone targeted site-specific recombination.

Another way of estimating the efficiency of the recombination may be carried our indirectly by immunohistochemistry, by analyzing the expression of the gene sequence to be inactivated for example.

According to a preferred embodiment, said fusion protein is encoded by a fusion gene integrated into one or more of the chromosomes of said cell of said organism. According to another embodiment, the fusion protein is encoded by a fusion gene integrated into an expression vector. The fusion gene according to the invention is introduced into the cell in the form of an expression vector or of one of its fragments. A "vector" is a replicon in which another polynucleotide segment (i.e. the fusion gene) is attached, so as to bring the replication and/or expression to the attached segment. The vector may be in particular a bacterial plasmid DNA, a cosmid, a phage DNA, a viral DNA or a minichromosome (BAC, YAC and the like). Such a vector may be integrative, that is to say can integrate into the genome of the host cell or can exist in the form of an extrachromosomal replicon. When it exists in the form of an extrachromosomal replicon, the expression vector is capable of replicating autonomously. When it is a fragment of an expression vector, preferably this fragment integrates into the cellular genome. The expression vector or one of its fragments comprises at least the fusion gene and a promoter or expression elements which make it possible to direct and control the expression of said fusion protein in at least one cell of said organism.

The expression vector comprises, in addition, signals for initiation and termination of the transcription, as well as appropriate regions for regulation of the transcription. These various control signals are chosen according to the cellular host used.

The expression elements controlling expression is understood to mean all the DNA sequences involved in the regulation of the gene expression, that is to say the minimal promoter sequence, the upstream sequences, the activating sequences ("enhancers"), optionally the inhibitory sequences ("silencers"), the "insulator" sequences, and any other required sequence.

Preferably, the fusion gene is placed under the control of tissue-specific or cell-specific or ubiquitous expression elements.

The tissue-specific expression elements or tissue-specific promoter regions are chosen from the promoters which make it possible to obtain a specific, and preferably high, expression in one or more cells, tissues, cell types or organs of the organism according to the invention. These promoter regions may be heterologous or nonheterologous to the organism and may be naturally present or otherwise in the genome of the organism. By way of nonlimiting example of tissue-specific promoter regions, there may be mentioned the promoter regions of the genes: for cytokeratin, and more particular for cytokeratin 5 (K5) and cytokeratin 14 (K14), which directs the expression of the gene in the basal keratinocytes of the epidermis; for .alpha.-1-antitrypsin which directs the expression of the gene in the hepatocytes; for the adipocyte fatty acid binding protein 2 (aP2) which directs the expression of the gene in the adipocytes.

According to a preferred embodiment, said organism is characterized in that said promoter region is the cytokeratin 5 (K5) promoter region and said fusion gene Cre-ER.sup.T.

According to a second preferred embodiment, said organism is characterized in that said promoter region is the cytokeratin 5 (K5) promoter region and said fusion gene Cre-ER.sup.T2.

According to a third preferred embodiment, said organism is characterized in that said promoter region is the cytokeratin 5 (K5) promoter region and said fusion gene Cre-ER.sup.T3.

According to a fourth preferred embodiment, said organism is characterized in that said promoter region is the cytokeretin 14 (K14) promoter region and said fusion gene Cre-ER.sup.T.

According to a fifth preferred embodiment, said organism is characterized in that said promoter region is the cytokeratin 14 (K14) promoter region and said fusion gene Cre-ER.sup.T2.

According to a sixth preferred embodiment, said organist is characterized in that said promoter region is the cytokeratin 14 (K14) promoter region and said fusion gene Cre-ER.sup.T3.

According to a seventh preferred embodiment, said organism is characterized in that said promoter region is the .alpha.-1-antitrypsin promoter region and said fusion gene Cre-ER.sup.T.

According to an eighth preferred embodiment, said organism is characterized in that said promoter region is the .alpha.-1-antitrypsin promoter region and said fusion gene Cre-ER.sup.T2.

According to a ninth preferred embodiment, said organism is characterized in that said promoter region is the .alpha.-1-antitrypsin promoter region and said fusion gene Cre-ER.sup.T3.

According to a tenth preferred embodiment, said organism is characterized in that said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER.sup.T.

According to an eleventh preferred embodiment, said organism is characterized in that said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER.sup.T2.

According to a twelfth preferred embodiment, said organism is characterized in that said promoter region is the adipocyte fatty acid binding protein 2 (aP2) promoter region and said fusion gene Cre-ER.sup.T3.

According to a first embodiment, the organism according to the invention is characterized in that said fusion gene has the sequence SEQ ID No. 3 and encodes the Cre-ER.sup.T protein having the sequence SEQ ID No. 4.

According to a second embodiment, the organism according to the invention is characterized in that said fusion gene encodes, of sequence SEQ ID No. 5 the fusion protein Cre-ER.sup.T2 having the sequence SEQ ID No. 6.

According to a third embodiment, the organism according to the invention is characterized in that said fusion gene encodes, of sequence SEQ ID No. 7 the fusion protein Cre-ER.sup.T3 having the sequence SEQ ID No. 8.

The article by Metzger and Feil (1999) gives by way of nonlimiting example (cf. table page 471) a list of tissue-specific promoter regions which are capable of being used to direct the expression of the Cre protein in various tissues.

The tissue-specific promoter regions are mare generally chosen from those which direct the expression of the fusion protein in a physiological system, an organ, a tissue, a cell type or a particular cell, among which there may be nonexhaustively mentioned the nervous system in general, and in particular the brain, the cerebellum, the neurons, the motoneurons, the glial cells, the Schwann cells, the hypophysis, the hypothalamus, the pituitary gland, the hippocampus and the cortex, the heart, the ventricular cardiomyocytes and the auricular cardiomyocytes, the lungs, the bones, the eyes, and more particularly the retina and the crystalline lens, the skin and more particularly the dermis and the epidermis, the muscles, and tore particularly the skeletal muscles, the cardiac muscle, the smooth muscles, the mammary gland, the gonads and more particularly the testes, the ovaries, the germ cells, the oocytes, the oogonias, the spermatozoa, the spermatogonias and the spermatocytes, the kidney, the liver and in particular the hepatocytes, the spleen, the pancreas and in particular the Langerhans' cells and the .beta. cells, the tongue, the esophagus, the adipocytes, the vascular endothelial cells.

The ubiquitous expression elements or ubiquitous promoter regions are chosen from the promoter regions which make it possible to obtain expression, preferably high expression, in all, or at least in a high proportion, of organs, or of tissues of the organism according to the invention. These promoter regions may be heterologous or nonheterologous to the organism according to the invention. By way of nonlimiting example of "so called" ubiquitous promoter regions, there may be mentioned the cytomegalovirus (CMV) promoter (Schmidt et al., 1990) and the interferon-inducible promoter (M.times.1) (Hug et al., 1998; Arnheiter et al., 1990). In addition, the expression elements, or promoter regions according to the invention, can ensure a constitutive or inducible Control of the expression of the fusion gene, Among the elements ensuring inducible expression, there may be mentioned the eukaryotic promoter regions which are inducible by heavy metals (Mayo et al., 1982; Brinster et al., 1982; Seark et al., 1985), by heat shock (Nover et al., 1991), by hormones (Lee et al., 1981; Hynes et al., 1981; Klock et al., 1987; Israel et al., 1989), by interferon (Hug et al ., 1998; Arnheiter et al., 1990). There may also be mentioned the inducible prokaryotic expression elements such as the E. coli Lac repressor system (LacR/operator/inducer) (Hu et al., 1987; Brown et al., 1987; Figge et al., 1988; Deuschle et al., 1990; Labow et al., 1990), the E. coli tetracycline resistance system (Gossen et al., 1992) (WO 94 04 672, EP 804 565).

In the case where the integration of the fusion gene is targeted by homologous recombination into the genome of the organism ("knock-in"), the fusion gene may be free of promoter regions or of expression elements and may be placed under the control of a promoter region or of endogenous expression elements.

The recombinant DNA technologies used for the construction of the expression vector according to the invention are those known and commonly used by persons skilled in the art. Standard techniques are used for cloning, isolation of DNA, amplification and purification; the enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases are carried out according to the manufacturer's recommendations. These techniques and others are generally carried out according to Sambrook et al. (1989).

The vector according to the invention or the vector fragments may be introduced into the host cell by standard methods such as for example microinjection into a pronucleus, transfection by calcium phosphate precipitation, lipofection, electroporation, heat shock.

The fusion gene according to the invention preferably comprises in the 5'.fwdarw.3' direction: a DNA fragment encoding the Cre recombinase of bacteriophage P1 or one of its variants; a DNA fragment of at least 45 nucleotides encoding at least either all or part of the D hinge region of a nuclear estrogen receptor, a region situated between the DNA-binding domain and the ligand-binding domain, or a peptide which is functionally equivalent to said D hinge region; and a DNA fragment encoding the ligand-binding domain (LBD) of a nuclear estrogen receptor or variants thereof, said fragment having at least one mutation conferring on LBD the capacity to respond to synthetic antiestrogens, but not to natural estrogenic agonists.

According to another embodiment of the invention, the fusion protein is directly introduced into the organism, or into a cell of the organism, it being possible for this introduction to be carried out by injection into a tissue or an organ in the case of an organism, or by microinjection in the case of a cell.

The DNA sequence of interest according to the invention is a gene or an intergenic sequence. According to a preferred embodiment of the invention, the DNA sequence of interest is a gene, it being possible for the function of the gene to be known or unknown. The study of an organism according to the invention exhibiting modification of a gene or of any other genomic region of unknown function makes it possible to contribute to the definition of the function of this gene or of this intergenic region. All the genes and intergenic regions of a metazoan organism are capable of being used in the context of the present invention; more particularly, there may be mentioned the RXR.sub..alpha., RXR.sub..beta., RXR.sub..gamma., RAR.sub..alpha., RAR.sub..beta., RAR.sub..gamma., SNF2.sub..beta. genes. The expression "DNA sequence of interest" naturally belonging to the genome of said organism, or DNA sequence of interest in its natural chromatin environment, is understood to mean an endogenous DNA sequence, such as an endogenous gene, present in the genome at its natural locus (loci).

According to a preferred embodiment of the invention, the organism according to the invention is an animal, in particular a mouse, characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER.sup.T having the sequence SEQ ID No. 4, or Cre-ER.sup.T2 having the sequence ID No. 6, or Cre-ER.sup.T3 having the sequence ID No. 8, said fusion gene being under the control of the cytokeratin K5 promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.

According to a second preferred embodiment of the invention, the organism according to the invention is characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER.sup.T having the sequence SEQ ID No. 4, or Cre-ER.sup.T2 having the sequence ID No. 6, or Cre-ER.sup.T3 having the sequence ID No. 8, said fusion gene being under the control of the cytokeratin K14 promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.

According to a third preferred embodiment of the invention, the organism according to the invention is characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER.sup.T having the sequence SEQ ID No. 4, or Cre-ER.sup.T2 having the sequence ID No. 6, or Cre-ER.sup.T3 having the sequence ID No. 8, said fusion gene being under the control of the adipocyte fatty acid binding protein 2 (aP2) promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.

According to a fourth preferred embodiment of the invention, the organism according to the invention is characterized in that at least one of the cells of said mouse comprises: a fusion gene encoding the fusion protein Cre-ER.sup.T having the sequence SEQ ID No. 4, or Cre-ER.sup.T2 having the sequence ID No. 6, or Cre-E.sup.T3 having the sequence ID No. 8, said fusion gene being under the control of the .alpha.-1-antitrypsin promoter; one or more chromosomal DNA sequences of interest in their natural chromatin context and flanked ("floxed") by a lox site.

The present invention also relates to methods of preparing a metazoan organism according to the invention.

A first method of preparation consists in the steps of: a) obtaining an embyronic stem (ES) cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequencers) of interest, located in one or more chromosomes, by homologous recombination; b) introducing said modified embryonic stem cell into an embryo of said organism; c) developing said embryo up to the stage of a fertile adult organism; d) crossing said fertile adult organism with a transgenic organism in which at least one of the cells expresses said fusion protein and obtaining the progeny derived from said crossing; and e) optionally, selecting, among said progeny, said metazoan organism.

A second method of preparation consists in the steps of: a) obtaining a somatic cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence(s) of interest, located in one or more chromosomes, by homologous recombination; b) transferring the nucleus of said modified somatic cell into the cytoplasm of an enucleated recipient oocyte; c) developing the embryo obtained in step b) up to the stage of a fertile adult organism; d) crossing said fertile adult organism with a transgenic organism in which at least one of the cells expresses said fusion protein and obtaining the progeny derived from said crossing; and e) optionally, selecting, among said progeny, said metazoan organism.

The expression transfer of the nucleus or nuclear transfer, for the purposes of the present invention, is understood to mean the transfer of nucleus of a vertebrate live donor cell, of an adult organism or at the fetal stage, into the cytoplasm of an enucleated recipient cell of the same species or of a different species. The transferred nucleus is reprogrammed to direct the development of the cloned embryos which may then be transferred into carrier females to produce the fetuses and the neonates, or used to produce cells of the internal cellular mass in culture. Various nuclear cloning techniques are capable of being used; among these, there may be mentioned those which have been the subject of patent applications WO 95 17500, WO 97 07668, WO 97 07669, WO 98 30683, WO 99 01163, WO 99 37143.

A third method of preparation consists in the steps of: a) obtaining an embyronic stem (ES) cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence(s) of interest, located in one or more chromosomes, by homologous recombination; b) introducing said modified embryonic stem cell into an embryo of said organism; c) developing said embryo; and d) introducing said fusion protein into at least one cell of said embryo or of the organism obtained from the development of said embryo.

A fourth method of preparation consists in the steps of: a) obtaining a somatic cell modified by insertion of site(s) of recognition for said recombinase protein into said DNA sequence(s) of interest, located in one or more chromosomes, by homologous recombination; b) transferring the nucleus of said modified somatic cell into the cytoplasm of an enucleated recipient oocyte; c) developing said embryo; and d) introducing said fusion protein into at least one cell of said embryo or of said organism obtained from the development of said embryo.

The insertion of the sites of recognition specific for the recombinase protein, in particular of the loxP site(s) for the Cre recombinase, into the DNA sequence of interest is preferably carried out by homologous recombination of the gene comprising said DNA fragment to be excised or inverted (two loxP sites) or respectively inserted or translocated (one loxP site) with a said modified gene comprising said DNA fragment to be excised flanked in 5' and/or 3' by said recombinase recognition site(s) according to the desired application, in particular the loxP sites.

To do this, the modified DNA fragment of interest may be integrated by homologous recombination into the genome of the cells of said organism before, at the same time, or after the step of introducing the fusion protein or of a transfer vector, or of a vector for expressing the fusion protein. Preferably, the DNA fragment of interest is introduced into pluripotent embryonic cells (ES cells) by the appropriate technique, such as for example electroporation, or the use of retrovitral vectors, calcium phosphate precipitation, lipofection.

The DNA constructs intended for homologous recombination will comprise at least a portion of the DNA sequence of interest, in particular of the gene or of the intergenic sequence of interest into which will be introduced the desired genetic modifications), such as the introduction of at least one recombinase recognition site, and which will include regions of homology with the target locus. For facilitated use, positive and/or negative selectable markers (for example the neo gene conferring resistance to the antibiotic G418) may be introduced. The selectable marker used to make it possible to identify the homologous recombination events may be disruptive, and may be eliminated, if necessary, if it is itself flanked by recombinase recognition sites such as the loxP (or FRT) sites. This makes it possible to obtain mice in which the sole modification at the level of the modified locus is the insertion of recognition sites such as loxP.

The metazoan organisms obtained by the methods of preparation presented above can then be treated with a synthetic ligand endowed with antiestrogenic activity such as Tam and OHT. In the various methods and uses of the invention, the bringing of said cells of said organism into contact with said synthetic ligand is carried out by administration by the oral or topical route, or by injection and in particular, by intravenous, intramuscular, intraspinal, intracerebral, intraperitoneal injection. In the case of an embryo, a fetus or a neonate before weaning, the treatment with the synthetic ligand may be carried out by administration to the mother. When this involves cells in culture derived from said organism, said syn