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

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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.