Vasquez K M, Marburger K, Intody Z, Wilson J H
Science Park Research Division, M. D. Anderson Cancer Center, Smithville, TX 78957, USA.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8403-10. doi: 10.1073/pnas.111009698.
Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies--chimeric nucleases and triplex-forming oligonucleotides--for stimulating recombination in cells.
基因打靶在哺乳动物细胞中已被证明在生物技术、基因结构与功能研究以及理解染色体动力学方面具有不可估量的价值。它还为基因治疗应用提供了一种潜在工具。当前技术存在两个限制因素：哺乳动物细胞中同源重组的低发生率以及载体DNA随机（非靶向）整合的高发生率。在此，我们在当前对重组机制和机器的理解框架内，考虑克服这些限制的可能方法。多项研究表明，通过过表达或干扰表达来瞬时改变重组蛋白的水平，或许能够增加同源重组或降低随机整合，我们列出了一系列候选基因。我们考虑对载体DNA进行潜在有益的修饰，并讨论DNA递送方法对打靶效率的影响。最后，我们展示了表明基因特异性DNA损伤可刺激局部同源重组的研究工作，并讨论了两种用于刺激细胞中重组的通用方法——嵌合核酸酶和三链形成寡核苷酸——的最新研究结果。
