Beta cell-specific ablation of target gene using Cre-loxP system in transgenic mice.
Beta cell-specific ablation of target gene using Cre-loxP system in transgenic mice.
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DOI：
10.1006/jsre.1999.5642
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发表时间：
1999-06
期刊：
影响因子：
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通讯作者：
M. K. Ray;S. P. Fagan;S. Moldovan;F. DeMayo;F. Brunicardi
中科院分区：
文献类型：
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作者：
M. K. Ray;S. P. Fagan;S. Moldovan;F. DeMayo;F. Brunicardi
使用Cre-loxP系统的基因的组织特异性失活已被用作定义其作用的重要工具，其中基因在每种细胞类型中的失活导致胚胎致死。Cre重组酶（Cre）的表达可以通过组织特异性启动子、配体诱导型启动子和配体依赖性Cre融合蛋白控制Cre表达的时间或空间分布来调节。大鼠胰岛素启动子（RIP）已在本研究中用于驱动Cre的表达，特别是在β细胞中。将Cre编码序列与RIP连接，并将分离的RIP-Cre转基因显微注射到一个细胞胚胎中以建立转基因小鼠系。用RIP-Cre转基因小鼠胰腺和其他组织的总RNA通过逆转录聚合酶链反应证明了大鼠胰岛素启动子的组织特异性。此外，通过将RIP-Cre转基因小鼠与携带β-肌动蛋白-loxP-CAT-loxP-lacZ转基因的报告小鼠杂交，进一步分析RIP的效率和特异性。在这些小鼠中，lacZ仅在通过Cre-介导的重组切除所述的CAT基因后表达。在这里，我们提供了双基因小鼠中lacZ的β细胞特异性表达的数据，作为靶向β细胞特异性基因的小鼠模型中的概念证明。RIP-Cre转基因小鼠将被用作靶向切除β细胞特异性基因的潜在工具，以研究它们在胰岛细胞生理学中的作用。
Tissue-specific inactivation of a gene using the Cre-loxP system has been used as an important tool to define its role in which the inactivation of the gene in every cell type results in an embryonic lethality. The expression of Cre recombinase (Cre) can be regulated by controlling the timing or spatial distribution of Cre expression via tissue-specific promoters, ligand-inducible promoters, and ligand-dependent Cre fusion proteins. The rat insulin promoter (RIP) has been used in this study to drive the expression of Cre, specifically in the beta cells. The Cre coding sequence was ligated with the RIP and the isolated RIP-Cre transgene was microinjected into one cell embryo to establish a transgenic mouse line. Tissue specificity of the rat insulin promoter was demonstrated by reverse transcriptase polymerase chain reaction using total RNA from pancreas and other tissues of the RIP-Cre transgenic mice. In addition, the efficiency and specificity of RIP was further analyzed by crossbreeding the RIP-Cre transgenic mice with reporter mice bearing a beta-actin-loxP-CAT-loxP-lacZ transgene. In these mice, lacZ is expressed only after excision of the floxed-CAT gene by Cre-mediated recombination. Here, we present the data for beta cell-specific expression of lacZ in the bigenic mice, as proof of concept in a mouse model for targeting beta cell-specific gene(s). The RIP-Cre transgenic mice will be used as a potential tool for targeting the excision of beta cell-specific gene(s) to study their role in islet cell physiology.