Understanding CLIM2 functions in vivo using transgenic RNAi

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DESCRIPTION (provided by applicant): Gene knockout technology in mice has been instrumental in investigating gene functions in mammals. It has been used to reveal gene functions in normal as well as in pathogenic pathways in vivo, and to generate disease models including models of many age-dependent diseases. However, the technical complexity, the length of time and the high cost of gene knockout approach has limited its wider use. This has slowed the analysis of gene functions in mammals. Recent experiments have demonstrated that transgenic RNAi can be a simpler, faster and more economical alternative for reverse genetics in mammals. We propose to test a more sophisticated transgenic RNAi strategy that will give investigator more control over where and when to induce gene silencing. We will test a construct that is driven by a ubiquitously active promoter, which synthesizes EGFP initially. This allows easy screen of transgenic lines that carry and express the transgene. The EGFP coding sequence is flanked with loxP site. Therefore, after exposure to Cre recombinase it will be excised, permitting the promoter to synthesize RFP and a pre-miRNA, which is placed in the 3'-UTR. The pre-miRNA can be processed in cells to form miRNA and mediate gene silencing. We propose to use this construct to express a miRNA that silences CLIM2 (cofactor of LIM homeodomain transcription factors, also known as Ldb1 or NLI), which is a cofactor of LIM homeodomain transcription factors (LIM-HD) and LIM-only oncoproteins (LMO). CLIM2 plays important roles in cell differentiation and patterning during development and is thought to play a role in cancer. However, functional studies of CLIM2 have been hampered by the early embryonic lethality of the knockout mice. Because gene silencing will be inducible spatially and temporally, our approach, if successful, will enable us to investigate the function of CLIM2 in cell differentiation, brain developmental patterning and cancer in detail. PUBLIC HEALTH RELEVANCE We will use a new method, transgenic RNAi, to determine CLIM2 gene functions in mammals. Compared with the existing method, the new method will be simpler, cheaper, faster and more flexible. If successful, our method will generate a mouse model for CLIM2 dysfunction, which will allow investigation into the role of CLIM2 in development and in cancer.