Gene Targeting Facility

基因打靶设施

• 批准号: 8763770
• 负责人: Lino Tessarollo
• 金额: $ 31.78万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8763770
• 关键词: Antibiotic Resistance; Blastocyst Transfer; CCR; Chimera organism; Color; Communities; Counseling; DNA; Development; Electroporation; Embryo; Engineering; Female; Fostering; Gene Targeting; Generations; Genes; Genetic Programming; Genets; Germ Lines; Goals; Implant; Injection of therapeutic agent; Modeling; Molecular; Molecular Biology; Mothers; Mus; Mutation; Neomycin; Pregnancy; Process; Protocols documentation; Puromycin; Reagent; Research Personnel; Staging; Technology; Thymidine Kinase; Time; Toxin; blastocyst; cancer genetics; court; design; embryonic stem cell; experience; functional genomics; homologous recombination; human disease; mouse genome; mouse model; offspring; pregnant; success; tool; vector

Genetically modified mice by means of homologous recombination are generated by injection of manipulated ES cells into recipient blastocysts. The injected blastocysts, following re-introduction into recipient foster mothers will produce chimeric mice in which the manipulated ES clones populate the germ line and transmit the desired mutation to the offspring. The technology to generate genetically modified chimeras involves 3 main sequential steps. 1- Engineering of the targeting vector to introduce the desired mutation into the mouse genome; 2- Introduction of the targeting vector into mouse embryonic stem cells (ES cells) to accomplish homologous recombination; 3- Injection with the targeted ES cells and immediate transfer of blastocysts into pseudo-pregnant recipient mothers. We provide diversified support to the CCR-NCI scientific community with counseling and technical help for all 3 different stages depending on the experience and needs of the investigator. 1-Engineering of the targeting vector. The generation of a targeting vector for homologous recombination in ES cells requires careful planning. It is of paramount importance for the overall success of a specific project that this step is well thought and planned. We provide scientific input for the designing of an optimal targeting vector. We make available to the investigators the best molecular tools to engineer the targeting vector including protocols and reagents for the use of the recombineering technology. Recombineering is a powerful tool that allows the generation of the desired DNA vectors in a relatively short period of time (Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet. 2001, 769-79). 2- Introduction of the targeting vector into the ES cells to accomplish homologous recombination. The targeting vector is introduced into mouse ES cells by electroporation. ES clones are positively selected for the presence of specific antibiotic resistance (for example neomycin, but also puromycin or blastycidin) and negatively by the presence of the Thymidine Kinase (TK) or Diphteria Toxin (DT) genes. Selected clones are then grown in duplicate and one set is given to the investigators for analysis of specific homologous recombination. 3- Injection of the targeted ES cells into mouse blastocysts and subsequent transfer into pseudo-pregnant recipient females. ES clones identified as correctly targeted are grown and expanded for the micro-injection into blastocysts at 3.5 days of gestation. The microinjected blastocysts are implanted into pseudo-pregnant recipient females who will generate chimeras derived from the blastocyst and the targeted ES clone. Coat color is used to score and identify the chimeras that will likely transmit the desired mutation to the progeny.

通过将操作的ES细胞注射到受体胚泡中来产生通过同源重组的遗传修饰小鼠。注射的胚泡在重新引入受体寄养母体后将产生嵌合小鼠，其中操纵的ES克隆填充生殖系并将所需的突变传递给后代。产生转基因嵌合体的技术包括3个主要的连续步骤。1-工程化靶向载体以将所需突变引入小鼠基因组; 2-将靶向载体引入小鼠胚胎干细胞（ES细胞）以实现同源重组; 3-注射靶向ES细胞并立即将胚泡转移到假孕受体母亲中。我们为CCR-NCI科学界提供多样化的支持，根据研究者的经验和需求，为所有3个不同阶段提供咨询和技术帮助。1-靶向载体的工程化。用于ES细胞中同源重组的靶向载体的产生需要仔细规划。这一步的周密考虑和计划对一个具体项目的全面成功至关重要。我们为最佳靶向载体的设计提供科学投入。我们为研究人员提供最好的分子工具来设计靶向载体，包括使用重组工程技术的方案和试剂。优化是一种强大的工具，其允许在相对短的时间内产生所需的DNA载体（Copeland NG，Jenkins NA，Court DL.基因工程：小鼠功能基因组学的强大新工具。自然遗传学2001，769-79）。2-将靶向载体引入ES细胞以实现同源重组。通过电穿孔将靶向载体引入小鼠ES细胞。ES克隆因特异性抗生素抗性（例如新霉素，还有嘌呤霉素或杀稻瘟菌素）的存在而被阳性选择，并且因胸苷激酶（TK）或白喉毒素（DT）基因的存在而被阴性选择。然后将选择的克隆一式两份生长，并将一组提供给研究者用于分析特异性同源重组。3-将靶向的ES细胞注射到小鼠胚泡中，随后转移到假怀孕的受体雌性中。使鉴定为正确靶向的ES克隆生长并扩增，用于在妊娠3.5天时显微注射到胚泡中。将显微注射的胚泡植入假孕受体雌性，其将产生源自胚泡和靶向ES克隆的嵌合体。毛色用于评分和鉴定可能将所需突变传递给后代的嵌合体。