Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors.
Targeted gene modification in mouse ES cells using integrase-defective lentiviral vectors.
复制标题
使用整合酶缺陷型慢病毒载体对小鼠 ES 细胞进行靶向基因修饰。
作者：
Okada Y;Ueshin Y;Hasuwa H;Takumi K;Okabe M;Ikawa M#
慢病毒载体有效地整合到分裂和非分裂细胞的宿主基因组中，因此它们已被用于生物学和生物医学研究中的稳定转基因表达。然而，最近的研究强调了插入突变和随后的肿瘤发生的风险。在这里，我们使用整合酶缺陷型慢病毒（IDLV）载体来减少随机整合的机会，并检查慢病毒载体介导的基因靶向小鼠胚胎干细胞（ES）的可行性。在用野生型慢病毒载体转导后，发现512个G418抗性克隆中没有一个是同源重组克隆。尽管IDLV载体的转导效率较低（野生型的5.9%），但在941个G418抗性克隆中的9个中观察到成功的同源重组（0.83 ± 1.32%）。同源重组ES细胞的多能性通过嵌合小鼠的产生和随后的生殖系传递来证实。由于慢病毒载体可以有效地扩增各种干细胞类型，我们的策略在治疗应用中对安全的基因操作具有潜在的相关性。创世纪47：217-223，2009。© 2009 Wiley利斯公司
Lentiviral vectors efficiently integrate into the host genome of both dividing and nondividing cells, and so they have been used for stable transgene expression in biological and biomedical studies. However, recent studies have highlighted the risk of insertional mutagenesis and subsequent oncogenesis. Here, we used an integrase‐defective lentiviral (IDLV) vector to decrease the chance of random integration and examined the feasibility of lentiviral vector‐mediated gene targeting into murine embryonic stem (ES) cells. After transduction with wild‐type lentiviral vectors, none of the 512 G418 resistant clones were found to be homologous recombinant clones. Although the transduction efficiency was lower with the IDLV vectors (5.9% of wild‐type), successful homologous recombination was observed in nine out of the 941 G418 resistant clones (0.83 ± 1.32%). Pluripotency of the homologous recombinant ES cells was confirmed by the production of chimeric mice and subsequent germ line transmission. Because lentiviral vectors can efficiently transduce a variety of stem cell types, our strategy has potential relevance for secure gene‐manipulation in therapeutic applications. genesis 47:217–223, 2009. © 2009 Wiley‐Liss, Inc.