Generation of Targeted Mutation in Mice

转基因小鼠通过同源重组的方式，通过将操纵的ES细胞注射到受体囊胚中而产生。注射的囊胚，在重新引入受体寄养母亲后，将产生嵌合小鼠，在其中，被操纵的ES克隆繁殖在生殖系中，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-改造靶向载体以将所需的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-将靶向ES细胞注射并立即将囊胚移植到假孕受体母亲体内。我们为CCR-NCI科学界提供多样化的支持，根据研究人员的经验和需求，为所有三个不同阶段提供咨询和技术帮助。1-靶向载体的工程化。在ES细胞中产生用于同源重组的靶向载体需要仔细的规划。对于一个具体项目的总体成功来说，这一步骤经过深思熟虑和计划是至关重要的。为设计最优靶向载体提供了科学依据。我们为研究人员提供了最好的分子工具来设计靶向载体，包括重组工程技术使用的方案和试剂。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：研究小鼠功能基因组学的有力新工具。纳特·雷夫·吉内特。2001,769-79)。2-将靶向载体导入ES细胞，实现同源重组。通过电穿孔将靶向载体导入小鼠ES细胞。ES克隆的阳性选择是因为存在特定的抗生素耐药性(例如，新霉素，但也包括腐霉素或杀菌素)，而阴性选择是因为存在胸苷激酶(TK)或白喉毒素(DT)基因。然后，选定的克隆被复制生长，并将一套克隆提供给研究人员用于特定同源重组的分析。3-将靶向ES细胞注射到小鼠的囊胚中，然后转移到假孕的受体雌性鼠体内。被确认为正确靶向的ES克隆被培养和扩大，以便在怀孕3.5天时显微注射到囊胚中。显微注射的胚泡被植入假孕受体雌性体内，她们将产生来自胚泡和靶向ES克隆的嵌合体。毛色被用来对嵌合体进行评分和鉴定，这些嵌合体可能会将所需的突变传递给后代。

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.