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改。再加上ES细胞技术， 基因打靶为全面反向遗传学开辟了道路 哺乳动物。基因打靶在解决根本问题方面具有巨大潜力 有关基因表达、发育、癌症、免疫等的问题 生理学，用于创建人类疾病的动物模型，以及 治疗人类遗传疾病。 自从首次靶向天然哺乳动物基因以来的很短的时间内 1985年证明几十个哺乳动物基因已成为目标。 然而，哺乳动物细胞中的基因靶向绝非易事， 主要是因为目标事件发生的频率非常低并且 被 100-10000 倍高频率的随机积分所掩盖 事件。 靶向不同基因的效率差异更大 比四个数量级。 该应用建议使用可选择的、特征良好的 APRT CHO 细胞中的基因，用于研究希望中靶向变异的基础 利用它来改善基因靶向。 几个潜力 将研究影响。 (1)重组基因序列。亚太RT 系统将用于以两种方式搜索重组序列： (i) 参与其他研究的潜在候选人将 被筛选； (ii) 直接选择刺激序列。 (2)双链断裂。 双链断裂的可能性 目标基因可能会刺激定向重组 通过将 H2O 核酸内切酶识别位点插入 APRT基因。 无论 HO 存在或不存在，都会进行目标定位 核酸内切酶。 (3) 转录状态。 内源性启动子 APRT基因将被替换为可调启动子以测量 不同转录水平对基因频率的影响 瞄准。 (4)重组基因。 一种新的遗传方法基于 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，这些研究将推进 对哺乳动物基因靶向的了解可能会导致改善 目标频率。