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Generation and Characterization of Knockout Mice Targeting REV7 gene, which is involved in DNA damage tolerance

靶向 REV7 基因的敲除小鼠的生成和表征，该基因参与 DNA 损伤耐受

基本信息

批准号：
17590340
负责人：
MURAKUMO Yoshiki
金额：
$ 2.24万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

In the present study, we aimed to generate and analyze REV7 knockout mice. We carried out the construction of a knockout vector targeting the middle region of REV7, exon 3 to 6, in which REV7 interacts various proteins. The knockout vector was introduced into ES cells, and the cells were incubated in the selection medium with G418. About 150 G418-resistant colonies were cloned, and their genomic DNAs were extracted and used for PCR screening to identify homologous recombinants. However, we could not find homologous recombinants. Then we constructed another knockout vector using a different plasmid vector and introduced the vector into ES cells. About 750 G418-resistant clones were isolated and screened for identification of homologous recombinants. Despite many times of screening using various PCR condition, we could not find homologous recombinants in these clones. Possible reasons for the failure to identify homologous recombinants were ; 1) the genomic region cloned into the knockout vectors might not be suitable for homologous recombination, 2) PCR screening was not appropriate for identification of homologous recombinants in this case. For this reason, we decided to make the other knockout construct again using the different region of REV7 genome for homologous recombination. We constructed a new knockout vector targeting the region from exon 1 to exon 6 of REV7. We will introduce the new vector into ES cells soon and advance our project to generate REV7 knockout mice.

在本研究中，我们的目的是产生和分析REV7基因敲除小鼠。我们构建了靶向REV7中间区域（外显子3至6）的敲除载体，其中REV7与各种蛋白质相互作用。将敲除载体引入ES细胞，并将细胞在具有G418的选择培养基中孵育。克隆了约150个G418抗性菌落，并提取其基因组DNA，用于PCR筛选以鉴定同源重组子。然而，我们没有找到同源重组体。然后，我们使用不同的质粒载体构建了另一个敲除载体，并将该载体导入ES细胞。约750个G418抗性克隆被分离和筛选用于鉴定同源重组体。尽管我们使用各种PCR条件进行了多次筛选，但在这些克隆中均未发现同源重组子。未能鉴定同源重组体的可能原因是：1）克隆到敲除载体中的基因组区域可能不适合同源重组，2）在这种情况下，PCR筛选不适合鉴定同源重组体。出于这个原因，我们决定使用REV7基因组的不同区域进行同源重组，再次制备另一个敲除构建体。我们构建了一个新的敲除载体，靶向REV7的外显子1至外显子6区域。我们将很快将新载体引入ES细胞，并推进我们的项目，以产生REV7敲除小鼠。