TARGETED RECOMBINATION

靶向重组

• 批准号: 2179211
• 负责人: JOHN H WILSON
• 金额: $ 19.1万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179211
• 关键词: CHO cells; L cell; adenine phosphoribosyltransferase; complementary DNA; cytogenetics; developmental genetics; disease /disorder model; embryonic stem cell; endonuclease; fungal genetics; gene conversion; gene expression; gene frequency; gene mutation; gene therapy; genetic manipulation; genetic mapping; genetic markers; genetic models; genetic promoter element; genetic recombination; genetic transcription; molecular cloning; nucleic acid sequence; protein biosynthesis; tissue /cell culture; transfection /expression vector; transposon /insertion element

Targeted recombination is a powerful tool for introducing precise modifications into mammalian genomes. Coupled with ES cell technology, gene targeting has opened the way for full scale reverse genetics in mammals. Gene targeting has enormous potential for answering fundamental questions about gene expression, development, cancer, immunity, and physiology, for creating animal models of human diseases, and for treating genetic diseases in humans. In the short time since targeting to a native mammalian gene was first demonstrated in 1985 several dozen mammalian genes have been targeted. Gene targeting in mammalian cells, however, is by no means easy, primarily because targeted events occur at a very low frequency and are obscured by a 100-10000 fold higher frequency of random integration events. The efficiency of targeting into different genes varies by more than four orders of magnitude. This application proposes to use the selectable, well characterized APRT gene in CHO cells to study the basis for targeting variability in hopes of harnessing it to improve gene targeting. Several potential influences will be studied. (1) RECOMBINOGENIC SEQUENCES. The APRT system will be used to search for recombinogenic sequences in two ways: (i) potential candidates that have been implicated in other studies will be screened; and (ii) stimulatory sequences will be directly selected. (2) DOUBLE-STRAND BREAKS. The possibility that double-strand breaks in the target gene may stimulate targeted recombination will be investigated by inserting the HO endonuclease recognition site into the APRT gene. Targeting will be in the presence and absence of the HO endonuclease. (3) TRANSCRIPTION STATUS. The promoter for the endogenous APRT gene will be replaced with a tunable promoter to measure the effects of different levels of transcription on the frequency of gene targeting. (4) RECOMBINATION GENES. A novel genetic approach based on protein-protein interactions in yeast will be used to isolate mammalian recombination genes. Collectively, these studies will advance understanding of mammalian gene targeting and may lead to improved targeting frequencies.

靶向重组是引入精确的强大工具 对哺乳动物基因组的修饰。加上ES细胞技术， 基因靶向为全尺度反向遗传学开辟了道路 哺乳动物。基因靶向具有巨大的回答基本潜力 有关基因表达，发育，癌症，免疫力和 生理学，用于创建人类疾病的动物模型以及 治疗人类的遗传疾病。 自靶向天然哺乳动物基因以来的短时间内首先 1985年证明了数十个哺乳动物基因。 然而，哺乳动物细胞中的基因靶向绝不是容易的， 主要是因为目标事件发生在非常低​​的频率，并且是 被100-10000倍的随机整合频率更高 事件。 靶向不同基因的效率通过更多 大于四个数量级。 该应用程序建议使用可选的，良好的特征APRT CHO细胞中的基因以研究靶向变异性的基础 利用它改善基因靶向。 几个潜力 将研究影响。 （1）重组序列。 aprt 系统将以两种方式用于搜索重组序列： （i）与其他研究有关的潜在候选人将 被筛选； （ii）将直接选择刺激序列。 （2）双链断裂。 双链破裂的可能性 靶基因可能会刺激靶向重组 通过将HO核酸内切酶识别地点插入 APRT基因。 定位将在HO的存在和不存在的情况下 核酸内切酶。 （3）转录状态。 内源性的启动子 APRT基因将用可调启动子代替，以测量 不同水平转录对基因频率的影响 定位。 （4）重组基因。 一种基于新颖的遗传方法 酵母中的蛋白质 - 蛋白质相互作用将用于分离哺乳动物 重组基因。 总的来说，这些研究将进步 了解哺乳动物基因靶向，并可能导致改善 定位频率。