TARGETED MUTATIONS OF TYROSINE KINASE GENES IN THE MOUSE

该项目将包括方法的发展选择 将逆转录病毒DNA整合到特定宿主基因中 以产生特定的突变，并针对逆转录病毒感染， 携带特定细胞表面蛋白的特定细胞类型。 在我们以前的工作中，我们已经证明了 通过插入失活X连锁的 小鼠次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因 （HGPRTase）。 我们建议扩展这些研究，以便 破坏其他更有趣的靶基因， 过程的效率。 我们将尝试选择 转化体由于隐性抑癌基因的破坏， 最近被证明是视网膜母细胞瘤系统的一部分。 另一 强有力的选择是细胞在诱导后的存活， 终末分化 我们还将尝试选择 将逆转录病毒基因组插入特别受调控的基因 方法;该程序类似于使用的Mud-lac基因融合 在细菌中。 最后，我们描述了提高效率的努力 逆转录病毒DNA靶向特定的宿主基因， 在病毒基因组中掺入潜在的 与目的基因同源。 在一个单独的子项目中，我们将 尝试将病毒定位到特定的细胞类型。 在其中一些 实验中，病毒的env基因将被部分破坏， 用编码金黄色葡萄球菌蛋白的基因取代 A. 将这些病毒暴露于特定的单克隆IgG 制备将用抗体包被病毒， 可变特异性决定域向外。 的能力 这些病毒进入并在表达抗原的细胞上生长 单克隆抗体识别的抗体将通过测量 感染后子代病毒的产生。

This project will consist of the development of methods to select for the integration of retroviral DNAs into particular host genes to generate specific mutations; and to target retroviral infection to particular cell types bearing specific cell-surface proteins. In our previous work, we have demonstrated the feasibility of generating mutations by insertional inactivation of the X-linked mouse gene encoding hypoxanthine-guanine phosphoribosyl transferase (HGPRTase). We propose to extend these studies to facilitate the disruption of other more interesting target genes, and to improve the efficiency of the process. We will attempt to select for transformants due to the disruption of recessive anti-oncogenes, recently demonstrated for the retinoblastoma system. Another powerful selection is survival of cells after induction toward terminal differentiation. We will also attempt to select for insertion of retroviral genomes into genes regulated in particular ways; this procedure is analogous to the Mud-lac gene fusions used in bacteria. Lastly, we describe efforts to improve the efficiency of the targeting of retroviral DNAs to particular host genes by the incorporation into the viral genome of potential regions of homology with the target gene. In a separate subproject, we will try to target virus to particular cell types. In some of these experiments the viral env gene will be partially disrupted and substituted with the gene encoding the Staphlococcus aureus protein A. Exposure of these virus to particular monoclonal IgG preparations will coat the virus with antibody, oriented with the variable specificity-determining domain outward. The ability of these viruses to enter and grow on cells expressing the antigen recognized by the monoclonal antibody will be tested by measuring the production of progeny virus after infection.