Gene Targeting Facility

基因打靶设施

• 批准号: 10703051
• 负责人: Lino Tessarollo
• 金额: $ 53.32万
• 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/10703051
• 关键词: Alleles; Antibiotic Resistance; Blastocyst Transfer; CCR; COVID-19; CRISPR/Cas technology; Chimera organism; Color; Communities; Counseling; Cytoplasm; DNA; Development; Disease; Electroporation; Embryo; Engineering; Enzymes; Evolution; Female; Fostering; Gene Targeting; Generations; Genes; Genets; Germ Lines; Goals; Guide RNA; Implant; Injections; Molecular; Molecular Biology; Mothers; Mus; Mutagenesis; Mutation; Neomycin; Point Mutation; Pregnancy; Process; Protocols documentation; Puromycin; Reagent; Research Personnel; Technology; Thymidine Kinase; Time; Toxin; base; blastocyst; cancer genetics; cancer type; court; design; embryonic stem cell; experience; functional genomics; homologous recombination; human disease; mouse genome; mouse model; offspring; pregnant; procedure cost; programs; 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. As indicated above we have also started working with the CRISPR/Cas9 technology, the new evolution of creating targeted mutations in mice. With the CRISPR/Cas9 technology mutations can be introduced into a single gene or multiple genes at the same time by injecting different guide RNAs into the cytoplasm. The enzyme Cas9 uses the guide RNA to zero in on target DNA, then edits the DNA to disrupt genes or insert a desired sequence. CRISPR/Cas9 technology is a way to accelerate the generation of targeted mutation mouse models and it is a more efficient and a less costly procedure than the traditional ES cell-based targeted mutagenesis. These tools are used by investigators studying numerous types of cancer and other diseases, including COVID-19.

通过同源重组的方法，通过将操纵的胚胎干细胞注射到受体囊胚中产生转基因小鼠。被注射的囊胚，在重新导入受体养母体内后，将产生嵌合小鼠，在嵌合小鼠中，经过操纵的胚胎干细胞克隆填充生殖系，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三个主要的连续步骤。1-对目标载体进行工程改造，将所需的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）实现同源重组；3-注射目标胚胎干细胞并立即将囊胚移植到假怀孕的受体母亲体内。我们为CCR-NCI科学界提供多样化的支持，根据研究者的经验和需求，在所有3个不同阶段提供咨询和技术帮助。目标载体的工程。胚胎干细胞同源重组靶载体的生成需要仔细规划。对于一个特定项目的整体成功来说，这一步是经过深思熟虑和计划的，这是至关重要的。为最优目标向量的设计提供了科学的输入。我们为研究人员提供了最好的分子工具来设计靶向载体，包括使用重组技术的方案和试剂。重组是一种强大的工具，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：小鼠功能基因组学的一个强大的新工具。医学学报，2001,769-79)。2-将靶向载体导入胚胎干细胞，完成同源重组。通过电穿孔将靶向载体导入小鼠胚胎干细胞。ES克隆对特定抗生素（例如新霉素，但也包括嘌呤霉素或囊虫素）的抗性具有阳性选择，对胸苷激酶（TK）或白喉毒素（DT）基因的抗性具有阴性选择。选择的克隆然后在两个培养和一组给研究人员分析特定的同源重组。3-将目标胚胎干细胞注射到小鼠囊胚中，随后转移到假怀孕的雌性受体。被确定为正确目标的胚胎干细胞克隆在妊娠3.5天生长和扩增，用于微注射到囊胚中。将微注射的囊胚植入假怀孕的雌性受体，雌性受体将产生囊胚和目标胚胎干细胞克隆的嵌合体。毛色被用来标记和识别嵌合体，这些嵌合体可能会将所需的突变传递给后代。如上所述，我们也开始使用CRISPR/Cas9技术，这是在小鼠中产生靶向突变的新进化。利用CRISPR/Cas9技术，通过向细胞质中注入不同的引导rna，可以将突变同时引入单个基因或多个基因中。Cas9酶使用向导RNA锁定目标DNA，然后编辑DNA以破坏基因或插入所需序列。CRISPR/Cas9技术是一种加速产生靶向突变小鼠模型的方法，它比传统的基于胚胎干细胞的靶向突变更有效，成本更低。研究人员使用这些工具研究多种类型的癌症和其他疾病，包括COVID-19。