Development of method for gene function analysis using lentiviral vectors

随着人类和小鼠基因组测序的完成，人们对有效的基因功能分析工具的兴趣增加了。慢病毒载体已被开发用于针对未分裂细胞的基因治疗。在本研究中，我们对慢病毒载体进行了多次修改，并建立了基础研究的方法。我们还与其他实验室合作，利用慢病毒载体分析了一些基因的功能。1.我们开发了第三代慢病毒载体，以提高安全性和转导效率。这种慢病毒载体已被证明能够有效地将基因转移到造血干细胞、静止的原代CD4^+或CD8^+T细胞、B细胞、NK细胞、巨噬细胞、人类白血病细胞和黑色素瘤细胞。利用慢病毒载体，分析了CD226(dNaM-1)基因在CD226(dNaM-1)在T细胞中的表达，在巨噬细胞中分析了嵌合免疫球蛋白T细胞受体基因在巨噬细胞中的表达，在树突状细胞中分析了gp34/OX40L基因。2.我们构建了cre-lox载体和tet诱导载体来控制转基因的表达。利用小干扰RNA表达载体，失活PPARγ基因、突变的BRAF基因、Skp-2基因等，为各基因的功能鉴定提供了便利。为了控制siRNA的表达，我们构建了含有四环素Hi启动子和四环素抑制因子基因的慢病毒载体。4.将cDNA文库引入慢病毒载体，建立了表达克隆系统。这个系统使我们能够识别使细胞对艾滋病毒诱导的细胞死亡具有抵抗力的基因。

With the complete sequencing of the human and mouse genomes, the interest in tools for efficient gene function analysis has increased. Lentiviral vectors have been developed for gene therapy targeting nondividing cells. In this study, we have made several modifications to lentiviral vectors and developed method for basic research. We have also analyzed function of some genes using lentiviral vectors in collaboration with other laboratories.1.We have developed a third generation lentiviral vector to increase safety and transduction efficiencies. This lentiviral vector has been shown to be capable of efficient gene transfer into hematopoietic stem cells, resting primary CD4^+ or CD8^+ T cells, B cells, NK cells, macrophages, human leukemia cells, and melanoma cells. Using lentiviral vectors, CD226 (DNAM-1) gene in CD4^+ naive T cells, Bcl-3 in macrophages, a chimeric immunoglobulin T cell receptor gene in T cells, and gp34/OX40L gene in dendritic cells have been analyzed.2.We have constructed cre-lox vectors and tet-inducible vectors to control transgene expression.3.We have developed siRNA(small interfering RNA) expressing lentiviral vectors for downregulating gene expression. Using siRNA expressing vectors, inactivation of PPARγ gene, mutated BRAF gene, Skp-2 gene, etc.facilitated the functional characterization of each gene. In order to control siRNA expression, we have constructed lentiviral vectors containing the tetracycline operator site in the Hi promoter and the tetracycline represser gene.4.We have introduced cDNA library into lentiviral vectors and established expression cloning system. This system allowed us to identify genes that render cells resistant to HIV-induced cell death.