Gene Targeting Facility

基因打靶设施

• 批准号: 10487241
• 负责人: Lino Tessarollo
• 金额: $ 54.62万
• 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/10487241
• 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.

通过将操纵的ES细胞注射到受体胚泡中，通过同源重组而通过同源重组产生了转基因的小鼠。注射后的胚泡后，在重新引入接受者寄养母亲后会产生嵌合小鼠，其中操纵ES克隆填充了种系并将所需的突变传递给后代。生成转基因嵌合体的技术涉及3个主要顺序步骤。 1-靶向载体的工程，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）以完成同源重组； 3-注射靶向ES细胞，并立即将胚泡转移到伪孕的受体母亲中。我们根据调查员的经验和需求，通过咨询和技术帮助为CCR-NCI科学界提供多样化的支持。目标向量的1工程。 ES细胞中同源重组的靶向载体的产生需要仔细计划。对于特定项目的整体成功而言，这一步骤经过精心思考和计划至关重要。我们为设计最佳靶向向量设计提供了科学输入。我们向研究人员提供了最佳分子工具，以设计靶向向量，包括使用重新组合技术的协议和试剂。重新组合是一种强大的工具，可在相对较短的时间内生成所需的DNA载体（Copeland Ng，Jen​​kins NA，Court dl。重新组合：小鼠功能基因组学的强大新工具。NATREV 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引入小鼠ES细胞中。 ES克隆被积极地选择特定的抗生素耐药性（例如新霉素，但也是嘌呤霉素或乳霉素），并通过胸苷激酶（TK）或​​双翅目毒素（DT）基因而负面。然后将选定的克隆重复生长，并将一组给予研究者，以分析特定的同源重组。 3-将靶向ES细胞注射到小鼠胚泡中，然后转移到伪孕的受体女性中。在妊娠3.5天的3.5天内将其生长并扩展为胚泡的ES克隆，并扩展为微注射。将显微注射的胚泡植入伪孕的受体女性中，这些雌性将产生源自胚泡和靶向ES克隆的嵌合体。外套颜色用于评分和识别可能将所需突变传递给后代的嵌合体。如上所述，我们还开始使用CRISPR/CAS9技术，这是在小鼠中创建靶向突变的新演变。使用CRISPR/CAS9技术突变可以同时将不同的指导RNA注入细胞质中，将其引入单个基因或多个基因。酶Cas9使用引导RNA在目标DNA上为零，然后编辑DNA以破坏基因或插入所需的序列。 CRISPR/CAS9技术是一种加速靶向突变模型的产生的方式，它比传统的基于ES基于ES的靶向诱变更有效且成本较低。研究人员使用了许多类型的癌症和其他疾病（包括Covid-19）的研究人员使用了这些工具。