GENE MANIPULATION CORE

基因操纵核心

• 批准号: 7699756
• 负责人: Christopher A. Walsh
• 金额: $ 19.2万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7699756
• 关键词: 129 Mouse; Adenosine A1 Receptor; Adenoviruses; Adult; Aliquot; Animal Hospitals; Animal Husbandry; Animals; Area; Arts; Atlases; Back; Bacteria; Bacterial Artificial Chromosomes; Behavioral; Binding; Biological Assay; Biological Preservation; Brain; Breeding; Categories; Cell Survival; Cells; Chimera organism; Chromosomes; Classification; Cleaved cell; Cloning; Cloning Vectors; Code; Collection; Color; Complex; Consult; Consultations; Cryopreservation; Culture Media; DNA; DNA analysis; Dependovirus; Detection; Development; Devices; Diploidy; Disease; Dominant-Negative Mutation; Drug resistance; ES Cell Line; Educational process of instructing; Embryo; Embryo Transfer; Endoribonucleases; Ensure; Environment; Equipment; Event; Exhibits; Experimental Designs; Facility Construction Funding Category; Female; Figs - dietary; Fostering; Freezing; Funding; Gene Expression; Gene Targeting; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Recombination; Genome; Genotype; Germ Cells; Germ Lines; Growth; Guidelines; Harvest; Hippocampus (Brain); Hormones; Horns; Housing; Human Resources; Hybrids; Implant; In Vitro; Individual; Infection; Information Resources; Injection of therapeutic agent; Institution; Journals; Karyotype; Knock-in Mouse; Knock-out; Laboratories; Laboratory culture; Lentivirus Vector; Letters; Liquid substance; Location; Maintenance; Mammalian Oviducts; Medical; Mental Retardation and Developmental Disabilities Research Centers; Messenger RNA; Methods; Microinjections; Mouse Strains; Mus; Mutate; Mutation; Natural regeneration; Nervous system structure; Neuraxis; Neurons; Nitrogen; Numbers; Operative Surgical Procedures; Partner in relationship; Patient currently pregnant; Pattern; Pediatric Hospitals; Personal Satisfaction; Pharmaceutical Preparations; Phenotype; Physiological; Polymerase Chain Reaction; Population; Pregnancy; Procedures; Process; Production; Proteins; Protocols documentation; Quality Control; RNA; RNA Interference; Range; Reagent; Receptor Gene; Recombinant DNA; Recycling; Research; Research Personnel; Research Project Grants; Resources; Retroviridae; Rodent; Safety; Sampling; Series; Services; Site; Small Interfering RNA; Staging; Standards of Weights and Measures; Technical Expertise; Techniques; Technology; Time; Tissues; Training; Transfection; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Uterus; Vagina; Viral Vector; Work; Writing; adeno-associated viral vector; animal breeding; animal colony; animal facility; animal resource; base; blastocyst; cost; data management; day; design; design and construction; desire; egg; embryo cell; embryo culture; embryo tissue; embryonic stem cell; endoribonuclease; experience; expression vector; falls; gene therapy; germ free condition; homologous recombination; implantation; insight; male; medical schools; member; mouse genome; mutant; natural Blastocyst Implantation; new technology; next generation; novel; pathogen; pluripotency; programs; promoter; protein expression; pup; recombinase; repaired; research study; small hairpin RNA; sperm cell; stem; success; tissue culture; tool; transgene expression; transmission process; vector; zygote

5.a.2. Overall Objective The overall objective of the Gene Manipulation Core is to provide all MRDDRC investigators an affordable quality-controlled service for the generation of genetically altered mouse lines. Centralization within the Core of the state-of-the-art procedures that are required to generate genetically altered mice results in a costeffective, quality-controlled generation of these novel mice lines for MRDDRC investigators. The two main approaches to generating genetically altered mice are briefly summarized below. These are followed by a description in the Specific Objective section of the specific procedures that are required for these approaches and that are offered by the Core. 5.a.2.1. Gene Targeting For gene targeting in mice, mouse lines are generated in which endogenous (wild-type) genes are either entirely deleted, replaced with different genes, or are otherwise mutated to generate "knock-out" and "knock-in" mice. In many cases, conditional targeting allows investigators to control the timing during development when a targeted mutation occurs and/or the specific tissues in which this mutation occurs. The analyses of the phenotypes of mice with targeted mutations provides insight into the function of endogenous genes during developmental and disease processes. Gene targeting exploits the pluripotency of mouse embryonic ES cells which, when injected into host mouse embryos, are capable of generating germ cells (sperm and eggs) that can pass their genetic content on to subsequent generations. ES cells are manipulated in culture to alter endogenous ES cell genes with specific mutations. These "targeted" ES cells are then injected into mouse embryos that are grown to fully mature mice. ES cells contribute to the development of the germ cells, and the mating of these mice result in the passage of the targeted mutation to the next generation of mice. At this point, a novel mouse line with the desired mutation is established in which the mutation can be stably transmitted across generations. ES cells with specific mutations are generated by homologous recombination in which the endogenous wildtype ES cell genes are replaced by mutated genes contained in targeting vectors (Fig. C.5.1). These targeting vectors include genes that confer resistance to drugs that bias for the survival of cells that have undergone homologous recombination. ES cells are transfected with the targeting constructs and treated with selection drugs to eliminate cells that have not undergone homologous recombination. After selection, visible colonies appear which are composed of clones of an original single cell that survived the drug selection. Individual colonies are isolated and grown to provide samples for storage as well as for DNA analysis. Clones are genotyped to verify those that have undergone the correct homologous recombination event (positive clones). The chromosome contents of these clones are analyzed (karyotype analysis) to identify clones with the correct number of mouse chromosomes (40) Positive ES clones with good karyotypes are microinjected into mouse embryonic day three and one half (E3.5) blastocysts (see Fig. C.5.2). The injected blastocysts are surgically implanted into recipient females and pregnancies are allowed to go to full term. Mice born following these procedures are chimeric (sometimes referred to as FO chimeras); their tissues are derived from both the host blastocyst cells and the injected ES cells. A typical procedure involves the injection of ES cells derived from an agouti coat color 129 mouse strain into black C57BL/6 blastocysts. Chimeras that have a high percentage of agouti color are likely candidates for having germ cells derived from the mutant ES cells. Therefore these animals are likely capable of transferring the mutation to the next generation (F1). The mating of the chimeric animals to generate non-chimeric F1 offspring with the desired mutation finalizes the establishment of a novel mouse line with the mutation. 5.a.2.2. Transgenics Transgenic mouse lines are those in which exogenous DMA (transgenes) are randomly integrated into the genome. Transgenes direct the expression of molecules that can disrupt normal development and disease processes. The analyses of the effects of these molecules give insight to their functions. Transgenes consist of promoter sequences and coding sequences that direct the expression of proteins or RNA molecules that inhibit specific gene expression (RNAi). A variety of different promoter sequences allow researchers to limit the expression of transgenes to specific tissues and to specific times during development. Some promoters direct abnormally high levels of expression. The expression of other promoters can be regulated in an on/off manner by the treatment of transgenic animals with specific drugs that direct the activity of the promoters. The coding sequences can encode proteins or RNAi molecules that are either wild-type, or mutant. Mutant proteins include those that are abnormally active as well as those that act as dominant negative suppressors of normal endogenous protein activity. Standard transgenic technology involves the microinjection of transgenic DMA sequences into the pronuclei of single-cell embryos. In a subset of the injected embryos, the injected DNA will stably insert into the mouse genome. This insertion is random and individual embryos will contain insertions of the DNA at different loci of the genome. Usually, only one location in the genome per embryo has an insertion. The location of the insertion greatly influences the expression of the transgene. While the promoter and other regulatory sequences within the transgene direct the expression of the transgene to a certain extent, the influence of the site of insertion on expression results in a degree of randomness in the expression patterns of standard transgenes. Thus, once a line has been established, expression of the transgene must be assessed. Following microinjection of DNA, the single-cell embryos are allowed to develop in vitro overnight. A typical injection results in 50-80% of the injected embryos developing to the two-cell stage. These embryos are implanted into foster females and the pregnancy is allowed to go to term. The litters of pups born are designated the FO generation. Individual FO mouse pups are genotyped to identify those that have retained the injected transgene. These FO animals are bred when mature to identify those that pass the transgene to the next generation (F1). Demonstration that a transgene is transmitted to the F1 generation and the assessment of the transgene expression level and pattern are the final steps in the establishment of a new transgenic line.

5.a.2.总体目标 基因操作核心的总体目标是为所有 MRDDRC 研究人员提供负担得起的 用于产生转基因小鼠品系的质量控制服务。核心内的集中化 产生基因改造小鼠所需的最先进程序的结果是具有成本效益的， 为 MRDDRC 研究人员质量控制地生成这些新型小鼠品系。 下面简要总结了产生基因改造小鼠的两种主要方法。这些都是 随后在“具体目标”部分中描述了这些所需的具体程序 方法和核心提供的方法。 5.a.2.1。基因打靶 为了在小鼠中进行基因靶向，产生的小鼠品系中的内源（野生型）基因是 完全删除，用不同的基因替换，或者以其他方式突变以产生“敲除”和“敲入” 老鼠。在许多情况下，条件靶向允许研究人员在开发过程中控制时机 发生靶向突变和/或发生该突变的特定组织。的分析 具有靶向突变的小鼠的表型提供了对内源基因在 发育和疾病过程。基因打靶利用小鼠胚胎 ES 细胞的多能性 当注射到宿主小鼠胚胎中时，能够产生生殖细胞（精子和卵子） 可以将其遗传内容传递给后代。 ES细胞在培养物中被操纵以改变 具有特定突变的内源性 ES 细胞基因。然后将这些“靶向”ES细胞注射到小鼠体内 胚胎发育成完全成熟的小鼠。 ES细胞有助于生殖细胞的发育，并且 这些小鼠的交配导致目标突变传递给下一代小鼠。在此刻， 建立了具有所需突变的新型小鼠品系，其中突变可以稳定传播 跨越几代人。 具有特定突变的ES细胞是通过同源重组产生的，其中内源性 野生型 ES 细胞基因被靶向载体中包含的突变基因取代（图 C.5.1）。这些 靶向载体包括赋予对药物有抗性的基因，这些药物会偏向于具有抗药性的细胞的生存。 发生同源重组。 ES 细胞用靶向构建体转染并用 选择消除细胞的药物 那些没有经历过的 同源重组。 选择后，可见菌落 出现由以下组成 原始单细胞的克隆 在药物选择中幸存下来。 单独的菌落被隔离 并培养以提供样品 用于存储以及 DNA 分析。克隆被基因分型 验证那些有 经历了正确的 同源重组 事件（阳性克隆）。这 这些染色体的含量 分析克隆（核型 分析）来识别克隆 正确的鼠标数量 染色体 (40) 将具有良好核型的阳性 ES 克隆显微注射到第三天半的小鼠胚胎中 (E3.5)囊胚（见图C.5.2）。注射的囊胚通过手术植入受体雌性体内， 允许怀孕至足月。按照这些程序出生的小鼠是嵌合的（有时 称为 FO 嵌合体）；它们的组织来源于宿主囊胚细胞和注射的 ES 细胞。典型的程序包括注射来自刺鼠毛色 129 品系小鼠的 ES 细胞 转化为黑色 C57BL/6 囊胚。具有高比例刺豚鼠颜色的嵌合体可能是 具有源自突变ES细胞的生殖细胞。因此这些动物很可能能够转移 突变到下一代（F1）。嵌合动物交配产生非嵌合F1 具有所需突变的后代最终建立了具有该突变的新型小鼠品系。 5.a.2.2。转基因技术 转基因小鼠系是那些含有外源 DMA 的小鼠系 （转基因）随机整合到基因组中。转基因 指导可以破坏正常的分子的表达 发育和疾病过程。的影响分析 这些分子可以深入了解它们的功能。 转基因由启动子序列和编码序列组成 指导蛋白质或RNA分子的表达，从而抑制 特定基因表达（RNAi）。多种不同的启动子 序列允许研究人员将转基因的表达限制在 特定的组织和发育过程中的特定时间。一些 启动子指导异常高水平的表达。这 其他启动子的表达可以开/关方式调节 通过用特定药物治疗转基因动物 发起人的活动。编码序列可以编码 野生型或突变型的蛋白质或 RNAi 分子。 突变蛋白包括异常活性的蛋白以及 那些作为正常的显性负性抑制因子 内源性蛋白质活性。 标准转基因技术涉及显微注射 将转基因 DMA 序列导入单细胞胚胎的原核中。 在注射胚胎的子集中，注射的 DNA 将稳定插入 进入小鼠基因组。这种插入是随机且个别的 胚胎将在不同基因座处插入DNA 基因组。通常，每个胚胎基因组中只有一个位置具有 插入。插入位置影响很大 转基因的表达。而发起人和其他监管机构 转基因内的序列指导转基因的表达 在一定程度上，插入位点对表达的影响 导致表达模式具有一定程度的随机性 标准转基因。因此，一旦建立了线路， 必须评估转基因的表达。 DNA显微注射后，单细胞胚胎被允许 体外培养过夜。典型的注射结果为 50-80% 注射的胚胎发育到双细胞阶段。这些 胚胎被植入寄养雌性体内并怀孕 允许继续任期。出生的幼崽被指定为 FO 一代。对个体 FO 小鼠幼崽进行基因分型以识别那些 保留了注射的转基因。这些 FO 动物是饲养的 当成熟时识别那些将转基因传递给下一个的 一代（F1）。证明转基因被传递到 F1代和转基因表达水平的评估 和模式是建立新的 转基因系。