Establishment of novel conditional gene targeting methods using Cre-loxP system

基因打靶技术的建立使我们能够在小鼠染色体DNA上的任何靶基因中引入突变，但由于许多基因在小鼠的发育阶段发挥作用，突变小鼠经常遭受胚胎死亡，因此很难分析这些基因在成年组织中的功能。条件性基因打靶就是为了绕过这个问题而发明的。在这项研究中，我们建立了在小鼠身上实现组织或细胞系特异性基因打靶的新技术。我们的系统是基于Cre重组酶及其识别序列loxP介导的DNA重组，利用传统的基因打靶技术将一对loxP位点引入到小鼠染色体DNA的靶基因中。一种转基因等位基因也被用来验证所建立的技术，该等位基因可以在Gre-loxP介导的重组中赋予LacZ表达。为了在小鼠特定的组织或细胞中特异性地表达Cre基因，我们开发了两个新的系统。首先，我们构建了携带Ore基因的重组腺病毒，其启动子强大且普遍存在。通过这种病毒的感染，我们可以在小鼠的肝、肺、皮肤、胰腺和肠道等器官中特异性地引入靶基因的突变。虽然基因失活的效率不是很高(0.1-3%)，但我们可以使用这种方法精确地调节基因失活的开始。作为一种替代方法，我们还建立了几个转基因小鼠系，每个系都可以在特定的小鼠谱系中表达Ore重组酶。在该系统中，角蛋白14(K14)启动子、浦肯野细胞特异性蛋白2(PCP2)启动子和嗅觉标记蛋白(OMP)基因分别成功地在表皮基底层细胞、小脑浦肯野细胞和嗅神经细胞中表达。

An establishment of a gene targeting technology allows us to introduce mutations in any target genes on mouse chromosomal DNA.However, since many genes function in developmental stages of mice, mutant mice often suffer embryonic lethality and, therefore, it is difficult to analyze functions of these genes in adult tissues. Conditional gene targeting was invented to circumvent this problem. In this study, we established novel technologies to achieve tissue or cell-lineage specific gene targeting in mice. Our system is based on DNA recombination mediated by Cre recombinase and its recognition sequences, loxP.A pair of loxP sites was introduced into target genes on mouse chromosomal DNA using conventional gene targeting technology. A transgenic allele, which may confer LacZ expression upon Gre-loxP mediated recombination, was also used to validate established technology in this study. To express Cre gene specifically in particular tissues or cells in mice, we developed two novel systems. First, we constructed a recombinant adenovirus carrying Ore gene driven by strong and ubiquitous promoter. By the infection of this virus, we could specifically introduce mutations in target genes in various organs of mice, such as liver, lung, skin, pancreas and intestinal tract. Although the efficiency of gene inactivation is not so high (0.1-3%), we could precisely regulate an onset of gene inactivation using this method. As an altemative way, we also established several lines of transgenic mice, each of which may express Ore recombinase in specific lineage of mice. In this system, promoters of keratin 14 (K14), Purkinje cell specific protein 2 (PCP2), and olfactory marker protein (OMP) genes were successfully used to express Ore gene in epidermal basal cells, cerebellar Purkinje cells and olfactory nerve cells, respectively.