VERTEBRATE GENE TARGETING--A MODEL SYSTEM

当DNA分子被引入哺乳动物细胞或细胞核时 非洲爪哇卵母细胞，可用于同源的主要途径 这种外源DNA的重组是非保守的。在这种情况下 在非洲爪哇卵母细胞中，已经有可能在一些 通过表征重组中间体和通过检查 注射改性基质的效果。因为它的机制是 重组包括通过5‘->3’切除引入的DNA 核酸外切酶在互补链退火前是必不可少的 底物DNA是线性的。该模型到基因的外推 哺乳动物细胞中的靶向方案表明， 与染色体位置的同源相互作用如此罕见，以至于 目标并没有被打破。建议进行实验，以证明 对于卵母细胞中的重组来说是惰性的环状DNA成为 在体内被卵裂激活的。然后将探索方法，以实现 允许在环状DNA中引入双链断裂 卵母细胞位于一个特定但随意选择的位置。这些试剂将是 基于单链寡核苷酸之间的同源相互作用 和大肠杆菌RecA蛋白启动的双链DNA。光激活 DNA修饰剂将被拴在这种寡核苷酸上，并 被引入到已经含有环状靶DNA的卵母细胞中。这个 原型试剂会携带补骨脂素，而补骨脂素的命运 我们将详细研究交叉链接，以确定它们是否 重组。除了尝试实现有针对性的切割外， 可能与卵母细胞重组反应相关的其他参数 将对基因操作实验进行调查。这些措施包括 序列同源性的长度和质量对效率的影响 重组与重组中序列错配的命运 产品。最后，关键实验将在非洲爪哇卵中进行， 它们具有更复杂的重组能力，类似于 哺乳动物细胞。如果成功，这些研究将允许有针对性的 高效地操纵哺乳动物基因组。这将是 对实验动物基因功能的遗传学研究很有用， 像小鼠一样，并将提高基因干预的可行性 对人类遗传性疾病的治疗。

When DNA molecules are introduced into mammalian cells or into the nuclei of Xenopus laevis oocytes, the major pathway available for homologous recombination of this exogenous DNA is a nonconservative one. In the case of Xenopus oocytes, it has been possible to describe this pathway in some detail by characterizing recombination intermediates and by examining the effects of injecting modified substrates. Because the mechanism of recombination involves resection of the introduced DNA by a 5'->3' exonuclease prior to annealing of complementary strands, it is essential that the substrate DNA be linear. Extrapolation of this model to gene targeting protocols in mammalian cells suggests that the reason that homologous interactions with chromosomal sites are so infrequent is that the target is not broken. Experiments are proposed to demonstrate that circular DNAs, which are inert for recombination in oocytes, become activated by cleavage in vivo. Then approaches will be explored that will allow the introduction of a double-strand break into circular DNA in oocytes at a specific, but arbitrarily chosen site. These reagents will be based on homologous interactions between single-stranded oligonucleotides and double-stranded DNA promoted by E. coli RecA protein. Light-activated DNA modifying agents will be tethered to such oligonucleotides and introduced into oocytes that already contain a circular target DNA. The prototype reagent will carry a psoralen, and the fate of psoralen crosslinks will be explored in detail to determine whether they are recombinagenic. In addition to attempting to achieve targeted cleavages, other parameters of the oocyte recombination reaction that may be relevant to gene manipulation experiments will be investigated. These include effects of the length and quality of sequence homology on the efficiency of recombination and the fate of sequence mismatches in recombination products. Finally, critical experiments will be performed in Xenopus eggs, which have a more complex range of recombination capabilities, similar to mammalian cells. If successful, these studies will permit targeted manipulations of mammalian genomes with high efficiency. This would be useful for genetic investigation of gene function in experimental animals, like mice, and would improve the feasibility of genetic intervention in the treatment of inherited diseases in humans.