RECA/OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS

RECA/寡核苷酸定向诱变

• 批准号: 2421808
• 负责人: ROBERT A DUBIN
• 金额: $ 8.9万
• 依托单位: PROFILE DIAGNOSTIC SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2421808
• 关键词: DNA repair; Escherichia coli; bacterial proteins; gene targeting; molecular cloning; oligonucleotides; protein engineering; protein purification; recombinant proteins; recombinase; reporter genes; site directed mutagenesis; transcription termination; transfection

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.

特异性改变细胞基因组的能力有着巨大的希望 用于基因治疗，治疗某些传染病， 人类遗传疾病动物模型的建立。我们将开发一个 靶向诱变的一般方法能够引入特异性的， 单个碱基对改变到活细胞中的任何基因中，使用E. coli RecA蛋白和合成的寡核苷酸。急诊杆菌RecA 蛋白质在细菌同源重组中起重要作用， DNA修复并促进RecA包被的， 单链DNA和同源双链DNA。在这里，一个修改后的 RecA（工程化以包含核定位信号）与 用一个短的诱变寡核苷酸， 与靶细胞基因中发现的差异。这种核蛋白 使用脂质体将复合物引入培养的细胞中，其中RecA 保护诱变寡核苷酸免于降解，将其导向 核，促进寡核苷酸的搜索和配对 与其同源基因，并启动链交换。失配 异源双链内的扭曲刺激DNA修复和基因 转化，导致稳定导入细胞基因的 寡核苷酸上存在的序列改变。第一阶段将审查 RecA/阿糖胞苷依赖性染色体外靶向修复 突变报告基因在人类和灵长类动物培养细胞。效率 将定性和定量地监测基因修复。 拟定商业应用： 本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。潜在 应用包括体内和离体人基因治疗、治疗性 对某些癌症和传染病的干预， 用于结构/功能蛋白质分析的细胞，以及 人类遗传疾病的动物模型。

项目成果

Aldolase C/zebrin gene regulation by prolactin during pregnancy and lactation.

妊娠和哺乳期间催乳素对醛缩酶 C/zebrin 基因的调节。

• DOI: 10.1385/endo:20:1-2:91
• 发表时间: 2003
• 期刊: Endocrine
• 影响因子: 3.7
• 作者: Matsuda,Manabu;Lockefeer,JasonA;Horseman,NelsonD
• 通讯作者: Horseman,NelsonD

Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells.

在乳腺上皮细胞中通过 Jak2 和 Stat5a 诱导 NF-kappaB 配体的受体激活剂。

• DOI: 10.1074/jbc.m308545200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Srivastava,Sunil;Matsuda,Manabu;Hou,Zhaoyuan;Bailey,JasonP;Kitazawa,Riko;Herbst,MatthewP;Horseman,NelsonD
• 通讯作者: Horseman,NelsonD