Understanding CLIM2 functions in vivo using transgenic RNAi

描述（由申请人提供）：小鼠基因敲除技术有助于研究哺乳动物的基因功能。它已被用来揭示基因功能的正常以及在体内致病途径，并产生疾病模型，包括许多年龄依赖性疾病的模型。然而，基因敲除技术的复杂性、耗时长和成本高限制了其广泛应用。这减缓了对哺乳动物基因功能的分析。最近的实验表明，转基因RNAi可以是一种更简单，更快，更经济的替代哺乳动物的反向遗传学。我们建议测试一种更复杂的转基因RNAi策略，这将使研究人员能够更好地控制何时何地诱导基因沉默。我们将测试一个由普遍活跃的启动子驱动的构建体，该启动子最初合成EGFP。这允许容易地筛选携带和表达转基因的转基因系。EGFP编码序列侧翼为loxP位点。因此，在暴露于Cre重组酶后，它将被切除，允许启动子合成RFP和位于3 '-UTR中的pre-miRNA。pre-miRNA可以在细胞中加工以形成miRNA并介导基因沉默。我们建议使用这种构建体表达沉默CLIM 2（LIM同源结构域转录因子的辅因子，也称为Ldb 1或NLI）的miRNA，CLIM 2是LIM同源结构域转录因子（LIM-HD）和LIM-only癌蛋白（LMO）的辅因子。CLIM 2在发育过程中的细胞分化和模式化中起重要作用，并被认为在癌症中起作用。然而，CLIM 2的功能研究受到敲除小鼠早期胚胎致死性的阻碍。由于基因沉默在空间和时间上都是可诱导的，如果成功的话，我们的方法将使我们能够详细研究CLIM 2在细胞分化、大脑发育模式和癌症中的功能。我们将使用一种新的方法，转基因RNAi，以确定CLIM 2基因在哺乳动物中的功能。与现有方法相比，新方法将更简单，更便宜，更快速，更灵活。如果成功，我们的方法将产生CLIM 2功能障碍的小鼠模型，这将允许研究CLIM 2在发育和癌症中的作用。

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.