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.

通过同源重组产生的基因修饰小鼠是由 将操作的ES细胞注射到受体囊胚中。注射的胚泡， 在重新引入受体后，养母将产生嵌合小鼠，其中 操作的ES克隆填充生殖系并将所需突变传递给 后代产生转基因嵌合体的技术涉及3个主要顺序 步1-改造靶向载体以将所需突变引入小鼠 2-将靶向载体引入小鼠胚胎干细胞（ES细胞）， 3-用靶向ES细胞注射并立即进行同源重组; 将胚泡转移到假怀孕的受体母亲中。我们提供多样化的支持 向CCR-NCI科学界提供咨询和技术帮助， 这取决于研究者的经验和需求。1-工程学 目标载体用于ES中同源重组的靶向载体的产生 细胞需要仔细规划。这对一个国家的全面成功至关重要。 具体项目，这一步是经过深思熟虑和计划。我们提供科学投入， 最佳靶向载体的设计。我们为调查人员提供最好的 设计靶向载体的分子工具，包括使用的方案和试剂 重组工程技术。搜索是一个强大的工具，它允许生成 在相对较短的时间内将所需的DNA载体（Copeland NG，Jenkins NA， 法院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细胞中以实现 同源重组通过以下方式将靶向载体引入小鼠ES细胞中： 电穿孔ES克隆被阳性选择用于特异性抗生素的存在 抗性（例如新霉素，但也有嘌呤霉素或杀稻瘟菌素）和阴性的 存在胸苷激酶（TK）或白喉毒素（DT）基因。然后， 一式两份生长，并将一组交给研究人员进行特定分析 同源重组3-将靶向ES细胞注射到小鼠胚泡中， 随后转移到假怀孕受体雌性中。ES克隆鉴定为正确 在3.5天时，使靶向的细胞生长并扩增，用于显微注射到胚泡中。 怀孕将显微注射的胚泡植入假孕受体雌性中 他们将产生来自胚泡和靶向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 后代如上所述，我们也开始使用CRISPR/Cas9技术， 在小鼠中产生靶向突变的新进展。CRISPR/Cas9技术 通过注射，可以将突变同时引入单个基因或多个基因， 不同的引导RNA进入细胞质。Cas9酶利用向导RNA来定位 靶DNA，然后编辑DNA以破坏基因或插入所需序列。CRISPR/Cas9 技术是一种加速靶向突变小鼠模型生成的方法， 比传统的基于ES细胞的靶向治疗更有效， 诱变这些工具被研究人员用于研究多种类型的癌症和其他疾病。 疾病，包括COVID-19。