Loss of function through ablation of genes of importance in mice has been commonly employed in the field to generate animal model to study receptor and cpregulator function in diseases. However, many diseases are caused by gain of function through amplification or over-expression of coregulators and receptors. Thus, over expression of these genes in mice in a tissue specific manner will undoubtedly aid our understanding of the physiological function of these receptors. In this project, we propose to generate ES cells that can be used to over-express all 49 murine nuclear receptors and >10 coregulators relevant to metabolic diseases. The coregulators will include: PGC1a, SRC-3, SRC-2, SRC-1, RIP140 and other coregulators shown to be important for metabolic and cardiovascular diseases, cancer and aging. These ES cells can be: (1) used to produce mice over-expressing these factors in a tissue-specific manner in any tissue where specific Cre recombinase mouse line is available or (2) used to investigate the effect of their over-expression on stem cell function. The ES cells will be generated in a 'high throughput'fashion, since recombination frequency is very high in the ROSA locus. In addition, we will generate a limited number of adult mice from these ES cells for proof of principle. To generate knockin over-expressing ES cells, we will utilize a unique procedure. We have generated a targeting vector for the ROSA locus with a CAGGS promoter directing nuclear receptors and coregulators expression;a LoxP-Stop-LoxP cassette is inserted in between the CAGGS promoter and the coregulator/receptor coding sequence to prevent expression until recombination occurs. When mice harboring this knockin are crossed to tissue-specific Cre mice, the coregulator/receptor gene will be turned on and expressed in any tissue where the Cre is expressed. This system has many advantages: (1) no expression of gene of interest in mice in the absence of the Cre recombination;(2) a single mouse line can be used to overexpress coregulator/receptors in any given tissue of interest;(3) temporal expression can be effected if inducible expression of Cre is employed (tamoxifen or tetracycline inducible Cre);(4) ensure uniform expression level in any tissue of interest;(5) the locus/promoter limits expression of the inserted gene to no more than a few fold over the endogenous level;(6) in a similar token dominant negatives of regulatory molecules 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 upon request. In addition, easily used targeting cassettes will be made available for investigators in any field.
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