Loss of function through ablation of genes of importance in mice has been commonly employed in thefield to generate animal model to study receptor and cpregulator function in diseases. However, manydiseases are caused by gain of function through amplification or over-expression of coregulators andreceptors. Thus, over expression of these genes in mice in a tissue specific manner will undoubtedly aid ourunderstanding of the physiological function of these receptors. In this project, we propose to generate ES cellsthat can be used to over-express all 49 murine nuclear receptors and >10 coregulators relevant to metabolicdiseases. The coregulators will include: PGC1a, SRC-3, SRC-2, SRC-1, RIP140 and other coregulators shownto be important for metabolic and cardiovascular diseases, cancer and aging. These ES cells can be: (1) usedto produce mice over-expressing these factors in a tissue-specific manner in any tissue where specific Crerecombinase mouse line is available or (2) used to investigate the effect of their over-expression on stem cellfunction. The ES cells will be generated in a 'high throughput' fashion, since recombination frequency is veryhigh in the ROSA locus. In addition, we will generate a limited number of adult mice from these ES cells forproof of principle.To generate knockin over-expressing ES cells, we will utilize a unique procedure. We have generated atargeting vector for the ROSA locus with a CAGGS promoter directing nuclear receptors and coregulatorsexpression; a LoxP-Stop-LoxP cassette is inserted in between the CAGGS promoter and thecoregulator/receptor coding sequence to prevent expression until recombination occurs. When mice harboringthis knockin are crossed to tissue-specific Cre mice, the coregulator/receptor gene will be turned on andexpressed in any tissue where the Cre is expressed. This system has many advantages: (1) no expression ofgene of interest in mice in the absence of the Cre recombination; (2) a single mouse line can be used to overexpresscoregulator/receptors in any given tissue of interest; (3) temporal expression can be effected ifinducible expression of Cre is employed (tamoxifen or tetracycline inducible Cre); (4) ensure uniformexpression level in any tissue of interest; (5) the locus/promoter limits expression of the inserted gene to nomore than a few fold over the endogenous level; (6) in a similar token dominant negatives of regulatorymolecules can be expressed in specific tissues.Once ES cells are generated, frozen ES cells will be made available to all investigators in the field.Similarly, embryos generated from the mice we produced will also be freely distributed to investigator uponrequest. In addition, easily used targeting cassettes will be made available for investigators in any field.
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