A transgenic mouse, useful in screening for medicaments for the treatment of e.g., diabetes or skin cancers, comprises a fusion protein between a recombinase Cre, and a modified ligand binding domain of the nuclear estrogen receptor alpha

摘要

A metazoan organism (I), with the exception of humans, containing one fusion protein comprising a recombinase protein, the ligand binding domain of a vertebrate nuclear estrogen receptor and one or more DNA sequences of interest of (I), is new. A metazoan organism (I), with the exception of humans, where one cell of the organism comprises: (i) one fusion protein comprising a recombinase protein; a hinge region of at least 15 amino acids; and a polypeptide comprising the ligand-binding domain of the human or vertebrate nuclear estrogen receptor, their natural variants or one of their fragments, where the polypeptide exhibits at least one mutation relative to the wild-type form of the ligand-binding domain, or of their natural variants, or of their fragments. The fusion protein has negligible recombinase activity in the presence of a natural ligand, where recombinase activity is induced by small quantities of synthetic ligand endowed with antiestrogenic activity; and (ii) one or more genes or intergenic DNA sequences of interest naturally belonging to the genome of (I) into which one or more recognition sites of the recombinase protein are inserted, where the DNA sequences are located in one or more of the chromosomes of the genome of the cell. Independent claims are also included for the following: (1) preparation (M1) of (I) comprising: (a) obtaining an embryonic stem (ES) cell modified by insertion of site(s) of recognition for the recombinase protein into DNA sequences of interest, located in one or more chromosomes, by homologous recombination; (b) introducing the modified ES into an embryo of (I); (c) developing the embryo up to the stage of a fertile adult organism; (d) crossing the fertile adult organism with a transgenic organism in which at least one of the cells expresses the fusion protein, and obtaining the progeny derived from the crossing; and (e) selecting (I) from amongst the progeny (I). (2) a method (M2) of conditional recombination, especially excision, insertion, inversion, translocation, of the DNA sequence of interest where one or more of the chromosomes of the genome of the cell are inserted comprises: (a) bringing one cell of (I) into contact with a synthetic ligand endowed with antiestrogenic activity; (b) inducing the activity of the recombinase of fusion protein by the synthetic ligand. (3) obtaining (M4) (I), with the exception of humans, in which one cell possess an allele of a gene of interest inactivated by M2 ,and in which the other allele of the gene of interest possess a mutation, in exon and/or regulatory sequences, to obtain somatic mutations controlled in space and time and which are limited (point mutations, small deletions or insertion) in exon and/or regulatory sequences comprises: (a) obtaining (I) in which one cell of the germ line comprises a mutation in one of the alleles of the gene of interest; (b) crossing an organism obtained in (a) with (I); (c) selecting a progeny whose genome comprises a gene of interest in which one of the alleles possess a mutation and the other allele possesses at least two recombinase protein recognition sites oriented as a direct repeat; (d) using (M2) to conditionally delete the DNA fragment of the allele of the gene of interest which is flanked by two recombinase protein recognition sites oriented as a direct repeat; and (e) obtaining (I) in which the genome of one cell comprises gene of interest in which one allele is inactivated, while the other allele possess a somatic, preferably limited, mutation and preferably, in exon and/or regulatory sequences; (4) analyzing (M5) or studying the biological function of a DNA sequence of interest, especially a gene comprises: (a) bringing (I) or cells isolated from (I) into contact with a synthetic ligand endowed with antiestrogenic activity; (b) optionally inducing the expression of the fusion protein; (c) revealing the recombination event catalyzed by the recombinase activity of fusion protein; and (d) carrying out a biochemical, physiological, phenotypic, or behavioral study or analysis of a cell of (I); (5) screening (M6) compounds capable of being used as a medicament for the preventative and/or curative treatment of pathological conditions associated with alteration of the expression and/or function of DNA sequence of interest, comprises administering the compound to (I); (6) transgenic mice (II) K5-Cre-ERT/RXR alpha L2/L2, K5-Cre-ERT2/RXR alpha L2/L2, K5-Cre-ERT3/RXR alpha L2/L2, K14-Cre-ERT/RXR alpha L2/L2, K14-Cre-ERT2/RXR alpha L2/L2, K14-Cre-ERT3/RXR alpha L2/L2, whose RXR alpha gene may be selectively inactivated in the basal keratinocytes of the epidermis using (M2) following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in mouse alopecia and/or hyperproliferation of the basal keratinocytes and/or inflammatory reaction of the skin; (7) transgenic mice (III) alpha AT-Cre-ERT/RXR alpha L2/L2, alpha AT-Cre-ERT2/RXR alpha L2/L2, alpha AT-Cre-ERT3/RXR alpha L2/L2, whose RXR alpha gene may be selectively inactivated in the hepatocytes using M2 following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in mouse, an alteration of the proliferation of the hepatocytes; and (8) transgenic mice (IV) alpha P2-Cre-ERT/RXR alpha L2/L2, alpha P2-Cre-ERT2/RXR alpha L2/L2, alpha P2-Cre-ERT3/RXR alpha L2/L2, whose RXR alpha gene may be selectively inactivated in the adipocytes using M2 following treatment with a synthetic ligand endowed with antiestrogenic activity, causing in mouse, a alteration of the metabolism of lipids in the adipocytes and/or diabetes.