DNA REPAIR AND SOMATIC MUTATION IN ANTIBODY VARIABLE GENES

抗体可变基因中的 DNA 修复和体细胞突变

• 批准号: 2447741
• 负责人: P GEARHART
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2447741
• 关键词: DNA repair; gel electrophoresis; gene expression; gene mutation; genetically modified animals; hybridomas; laboratory mouse; molecular cloning; nucleic acid sequence; polymerase chain reaction; restriction endonucleases; southern blotting; ultraviolet radiation

A correlation between a genetic defect in DNA repair and lack of hypermutation would be very significant, since there are no in vivo models to study mutation. Three knockout lines of mice have been obtained: PMS2 and MSH2 mismatch repair mice, and XPA nucleotide excision repair mice. Homozygous (-/-) knockout and littermate (+/+) mice are being immunized, and DNA will be prepared from B cells. Endogenous Vx and VH genes are being amplified by PCR, cloned, and sequenced. The frequency of mutation in the V and constant genes from knockout and control mice are compared. Preliminary results show different patterns of mutation in normal and mismatch repair deficient mice. Hybridoma cells were irradiated with ultraviolet light. The cells were then grown in vitro and harvested at different time points. DNA was prepared, and incubated with restriction enzymes and T4 endonuclease, which cleaves thymine dimers. Cleaved DNA was separated by gel electrophoresis, and different genes were identified by Southern blot analysis. Current results suggest that V genes have a slower rate of repair than the dihydrofolate reductase gene.

DNA修复中的遗传缺陷与缺乏 超突变将是非常显著，因为在体内没有 研究突变的模型。 已经获得了三个敲除小鼠系：PMS 2和MSH 2 错配修复小鼠和XPA核苷酸切除修复小鼠。 免疫纯合（-/-）敲除和同窝（+/+）小鼠， DNA将从B细胞制备。 内源性Vx和VH基因是 通过PCR扩增、克隆和测序。 的频率 来自敲除小鼠和对照小鼠的V和恒定基因的突变是 比较了 初步结果显示， 正常和错配修复缺陷小鼠。 用紫外线照射杂交瘤细胞。 将细胞 然后在体外生长并在不同时间点收获。 DNA被 制备，并与限制性内切酶和T4核酸内切酶一起孵育， 其切割胸腺嘧啶二聚体。 裂解的DNA经凝胶电泳分离， 电泳，Southern杂交鉴定差异基因 分析. 目前的研究结果表明，V基因具有较慢的表达速率， 修复比二氢叶酸还原酶基因。