Gene Targeting Facility

基因打靶设施

基本信息

批准号：
8938475
负责人：
Lino Tessarollo
金额：
$ 32.98万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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.

通过同源重组的方式进行基因改造的小鼠是通过将经过操作的 ES 细胞注射到受体囊胚中而产生的。注射的囊胚在重新引入受体养母体内后将产生嵌合小鼠，其中经过操纵的 ES 克隆填充种系并将所需的突变传递给后代。产生转基因嵌合体的技术涉及 3 个主要的连续步骤。 1- 工程化靶向载体，将所需突变引入小鼠基因组； 2- 将靶向载体导入小鼠胚胎干细胞（ES细胞）中，完成同源重组； 3- 注射目标 ES 细胞并立即将囊胚移植到假孕受体母亲体内。我们根据研究者的经验和需求，为 CCR-NCI 科学界提供多元化支持，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。在 ES 细胞中生成用于同源重组的靶向载体需要仔细规划。这一步经过深思熟虑和规划对于特定项目的整体成功至关重要。我们为设计最佳靶向载体提供科学投入。我们为研究人员提供设计靶向载体的最佳分子工具，包括使用重组工程技术的方案和试剂。重组工程是一种强大的工具，可以在相对较短的时间内生成所需的 DNA 载体（Copeland NG、Jenkins NA、Court DL。重组工程：小鼠功能基因组学的强大新工具。Nat Rev Genet. 2001, 769- 79）。 2-将靶向载体引入ES细胞以完成同源重组。通过电穿孔将靶向载体导入小鼠 ES 细胞。 ES 克隆因特定抗生素抗性（例如新霉素、嘌呤霉素或稻瘟素）的存在而被正向选择，而胸苷激酶 (TK) 或白喉毒素 (DT) 基因的存在则被负向选择。然后将选定的克隆一式两份培养，并将一组提供给研究人员用于分析特定的同源重组。 3- 将靶向 ES 细胞注射到小鼠囊胚中，然后转移到假孕受体雌性体内。被确定为正确靶向的 ES 克隆会生长并扩增，以便在妊娠 3.5 天时显微注射到囊胚中。显微注射的囊胚被植入假怀孕的受体雌性体内，雌性受体将产生来自囊胚和目标 ES 克隆的嵌合体。毛色用于评分和识别可能将所需突变传递给后代的嵌合体。