The Establishment of Restriction-Modification Systems in Mammalian Cells

哺乳动物细胞限制性修饰系统的建立

• 批准号: 8700313
• 负责人: Thomas Gingeras
• 金额: $ 24万
• 依托单位: La Jolla Biological Laboratories
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8700313&HistoricalAwards=false
• 关键词: Establishment; Restriction; Modification; Systems; Mammalian

It has been demonstrated that it is possible to express site-specific bacterial methylase and endonuclease genes in eukaryotic cells. In addition the expression of these genes appears to have no deleterious effect on the host cell. In initial studies to express the PaeR7 methylase in mouse L cells, the levels of methylase produced varied greatly and were insufficient to protect the host cell genomes from the activities of the cognate endonucleases. The problems that need to be solved now in this proposal, are (1) the incomplete methylation of cell genome by PaeR7 and (2) the inefficient restriction of infecting viral DNA. In order to increase methylase and endonuclease activity, methods such as multiple rounds of transformation, selection of alternative promoters, gene amplification will be tried. Hopefully, a stable expression of the bacterial restriction/modification system in animal cells will be achieved. The ultimate goal of the project is to establish this system in cultured mammalian cells which would render them more resistant to viral infection. Dr. Gingeras is eminently qualified to carry out this work. Support if possible with a very good priority.

已经证明，在真核细胞中表达位点特异性细菌甲基化酶和核酸内切酶基因是可能的。此外，这些基因的表达似乎对宿主细胞没有有害影响。在小鼠L细胞中表达PaeR7甲基化酶的初步研究中，产生的甲基化酶水平变化很大，不足以保护宿主细胞基因组免受同源内切酶活性的影响。目前该方案需要解决的问题是：(1)PaeR7对细胞基因组的不完全甲基化；(2)对感染病毒DNA的低效限制。为了提高甲基化酶和核酸内切酶的活性，将尝试多轮转化、选择替代启动子、基因扩增等方法。希望能够在动物细胞中稳定表达细菌限制性修饰系统。该项目的最终目标是在培养的哺乳动物细胞中建立这个系统，使它们更能抵抗病毒感染。金格拉斯博士完全有资格从事这项工作。如果可能的话，优先提供支持。