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

基因靶向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。然而，由于许多基因在小鼠的发育阶段起作用，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。条件基因靶向的发明就是为了规避这个问题。在这项研究中，我们建立了在小鼠中实现组织或细胞系特异性基因靶向的新技术。该系统基于Cre重组酶及其识别序列loxP介导的DNA重组。利用常规基因靶向技术将一对loxP位点导入小鼠染色体DNA上的靶基因。在本研究中，还使用了一个转基因等位基因来验证已建立的技术，该等位基因可能在greg - loxp介导的重组中赋予LacZ表达。为了在小鼠的特定组织或细胞中特异性表达Cre基因，我们开发了两个新的系统。首先，我们构建了一种携带Ore基因的重组腺病毒，该重组腺病毒由强大且普遍存在的启动子驱动。通过这种病毒的感染，我们可以在小鼠的各个器官，如肝脏、肺、皮肤、胰腺和肠道中特异性地引入靶基因的突变。虽然基因失活的效率不高（0.1-3%），但我们可以通过这种方法精确地调控基因失活的起始时间。作为一种替代方法，我们还建立了几个转基因小鼠系，每个系都可以在小鼠的特定谱系中表达Ore重组酶。在该系统中，分别利用角蛋白14 （K14）、浦肯野细胞特异性蛋白2 （PCP2）和嗅觉标记蛋白（OMP）基因启动子在表皮基底细胞、小脑浦肯野细胞和嗅觉神经细胞中成功表达Ore基因。

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.