RECA/OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS

特定改变细胞基因组的能力大有可为。 用于基因治疗，某些传染病的治疗，以及 建立人类遗传病的动物模型。我们将开发一种 靶向突变的一般方法能够引入特定的， 用E。 ColiRecA蛋白和一种人工合成的寡核苷酸。大肠埃希氏菌RecA 蛋白质在细菌同源重组和 DNA修复和促进配对和链侵袭之间的RecA涂层， 体外单链DNA和同源双链DNA。在这里，一个经过修改的 RecA(设计为包含核定位信号)是复杂的 带有含有单一碱基的短致突变寡核苷酸 与在目标细胞基因中发现的不同。这种核蛋白 通过脂质体将复合体引入培养细胞，其中RecA 保护诱变寡核苷酸不被降解，将其引向 核，促进寡核苷酸的搜索和配对 与其同源基因结合，并启动链交换。不匹配 异源双链内的扭曲刺激DNA修复和基因 转化，导致稳定的导入到细胞基因中 寡核苷酸上存在序列改变。第一阶段将研究 依赖RecA/寡核苷酸的染色体外靶向修复 人和灵长类培养细胞中的突变报告基因。效率 将对基因修复进行定性和定量监测。 建议的商业应用： 这项研究旨在开发一种通用的方法 将特定的基因改变引入细胞。潜力 应用包括体内和体外人类基因治疗，治疗性 对某些癌症和传染病的干预、变更 用于结构/功能蛋白质分析的细胞，以及开发 人类遗传病的动物模型。

The ability to alter specifically the genome of cells holds great promise for gene therapy, treatment of certain infectious diseases, and the creation of animal models of human genetic disease. We will develop a general method of targeted mutagenesis capable of introducing a specific, single basepair alteration into any gene in a living cell using the E. coli RecA protein and a synthetic oligonucleotide. The E. coli RecA protein plays an essential role in bacterial homologous recombination and DNA repair and promotes pairing and strand invasion between RecA-coated, single-stranded DNA and homologous duplex DNA in vitro. Here, a modified RecA (engineered to contain a nuclear localization signal) is complexed with a short mutagenic oligonucleotide that contains a single base difference from that found in the target cellular gene. This nucleoprotein complex is introduced into cultured cells using liposomes, where RecA protects the mutagenic oligonucleotide from degradation, directs it toward the nucleus, promotes the searching and pairing of the oligonucleotide with its homologous gene, and initiates strand exchange. Mismatch distortion within the heteroduplex stimulates DNA repair and gene conversion, resulting in stable introduction into the cellular gene of the sequence alteration present on the oligonucleotide. Phase I will examine RecA/oligonucleotide-dependent targeted repair of an extrachromosomal mutant reporter gene in human and primate cultured cells. The efficiency of gene repair will be monitored qualitatively and quantitatively. PROPOSED COMMERCIAL APPLICATION: This research is directed towards developing a general method of introducing specific genetic alterations into cells. Potential applications include in vivo and ex vivo human gene therapy, therapeutic intervention for certain cancers and infectious diseases, alteration of cells for structural/functional protein analysis, and development of animal models for human genetic disease.