Development of novel gene trap vectors applicable to insertional mutagenesis in mice

插入诱变通常用于以系统方式产生突变体。胚胎干细胞的建立和基因捕获载体的发展，使插入突变技术用于突变小鼠的产生成为可能。因此，利用插入突变对小鼠进行系统性突变是包括我们在内的遗传学家目前研究的范围。虽然我们已经成功地建立了几个突变体与逆转录病毒基因陷阱载体在我们的实验室，我们也遇到了几个ploblems启动大规模诱变使用该载体。在本研究中，我们构建了两个新的载体，并使用这些载体启动了小规模的突变，作为大规模插入突变小鼠的可行性研究。我们一直在使用ROSA-BETAgeo，一种含有LacZ和neo融合基因的逆转录病毒载体，其5'端具有剪接受体序列。首先，我们将loxP序列插入ROSA-BETAgeo的3 'LTR，构建了一个新的载体ROSA-BETAgeo-loxP。当前病毒DNA整合到ES细胞染色体DNA上的基因中时，引入突变的基因陷阱机制的侧翼是一对loxP位点，因此，通过表达Ore重组酶而留下单个loxP位点，它很容易从染色体DNA中删除。因此，有可能用该系统产生可逆突变体。我们还产生了ROSA-IRES-BETAgeo，另一种在剪接受体序列和BETAgeo基因之间含有内部核糖体进入序列的新型载体。利用这种新的载体，我们分离了携带插入到目的基因3'侧内含子中的基因陷阱载体的突变体。因此，我们可以预期产生亚纯型突变体。利用本研究中产生的这些新型载体，我们可以建立20多个品系的突变小鼠。其中4个显示遭受已知基因的突变，2个品系显示胚胎致死性，表明在我们的系统中有效地产生突变体。

Insertional mutagenesis is commonly used to generate mutants in a systemic manner. Establishment of embryonic stem cells and development of gene trap vectors have made it possible to apply insertional mutagenesis for generation of mutant mice. Therefore, systematic mutagenesis of mice using insertional mutagenesis is currently in a scope of geneticists including us. Although several mutants have been successfully established with a retroviral gene-trap vector in our lab, we also encountered several ploblems to initiate a large scale mutagenesis using this vector. ln this study, we constructed two novel vectors and initiated a small scale mutagenesis using these vectors as a feasibility study for a large scale insertional mutagenesis of mice. We have been using ROSA-BETAgeo, a retrovirus vector containing a fusion gene of LacZ and neo, with splice acceptor sequences at its 5' end. First, we constructed a novelvector, ROSA-BETAgeo-loxP, by inserting a loxP sequence into 3'LTR of ROSA-BETAgeo. When provirus DNA integrated into a gene on chromosomal DNA of ES cells, a gene trap machinery, introducing a mutation, was flanked by a pair of loxP sites and, therefore, it was easily deleted from chromosomal DNA by expressing Ore recombinase leaving a single loxP site. Therefore, it is possible to generate reversible mutants with this system. We also generated ROSA-IRES-BETAgeo, another novel vector containing internal ribosome entry sequence in between splice acceptor sequence and BETAgeo gene. Using this novel vector, we isolated mutants carrying gene trap vectors inserted into introns at 3' side of target genes. Therefore, we could expect to generate hypomorphic mutants. Using these novel vectors generated in this study, we could establish more than 20 lines of mutant mice. Four of them were shown to suffer mutations of known genes and 2 lines showed embryonic lethality, suggesting efficient production of mutants in our system.