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 基因操纵核心的总体目标是为所有MRDDRC研究人员提供负担得起的 质量控制的服务，用于生成遗传变化的小鼠线。核心中的集中化 在产生遗传变化的小鼠所需的最新程序中，会导致成本效益， MRDDRC研究人员的这些新型小鼠系的质量控制生成。 在下面简要总结了产生遗传变化小鼠的两种主要方法。这些都是 然后在特定程序的特定目标部分中描述这些条件 方法是由核心提供的。 5.A.2.1。基因靶向 对于小鼠的基因靶向，产生了小鼠线，其中内源性（野生型）基因是 完全删除，被不同的基因取代，或者被突变以产生“敲除”和“敲门” 老鼠。在许多情况下，有条件的靶向允许调查人员控制开发过程中的时机 发生靶向突变和/或发生这种突变的特定组织。分析 具有靶向突变的小鼠的表型可洞悉内源基因在期间的功能 发育和疾病过程。基因靶向利用小鼠胚胎ES细胞的多能性 当注射到宿主小鼠胚胎中时，它能够产生生殖细胞（精子和卵） 可以将其遗传含量传递给后代。 ES细胞在培养中被操纵以改变 内源性ES细胞基因具有特异性突变。然后将这些“靶向” ES细胞注入小鼠 生长到完全成熟的小鼠的胚胎。 ES细胞有助于生殖细胞的发展， 这些小鼠的交配导致靶向突变通过下一代小鼠。在此刻， 建立了具有所需突变的新型小鼠系，可以稳定地传播突变 几代人。 具有特定突变的ES细胞是由同源重组产生的，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的抗性的基因 经过同源重组。 ES细胞用靶向构建体转染，并用 选择药物以消除细胞 没有经历的 同源重组。 选择后，可见菌落 出现由 原始单元的克隆 在选择药物中幸存下来。 单个菌落是孤立的 并成长以提供样品 用于存储和DNA 分析。克隆是基因型的 验证那些 经历了正确的 同源重组 事件（积极克隆）。这 这些染色体内容 分析克隆（核型 分析）以识别具有 正确的鼠标数 染色体（40） 带有良好核型的正ES克隆微注射到小鼠胚胎第三天半中 （E3.5）胚泡（见图C.5.2）。注射后的胚泡被手术植入受体女性和 允许怀孕完整。按照这些程序出生的小鼠是嵌合（有时 称为Fo Chimeras）；它们的组织均来自宿主胚泡细胞和注射的ES 细胞。一个典型的过程涉及注射源自Agouti Coat Color 129小鼠应变的ES细胞 进入黑色C57BL/6胚泡。具有高百分比Agouti颜色的嵌合体可能是候选人 具有源自突变体细胞的生殖细胞。因此，这些动物可能能够转移 下一代的突变（F1）。嵌合动物的交配生成非智力F1 带有所需突变的后代最终确定了具有突变的新型小鼠系。 5.A.2.2。转基因 转基因小鼠线是外源DMA （转基因）随机整合到基因组中。转基因 指导可能破坏正常的分子的表达 发展和疾病过程。对影响的分析 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 指导抑制蛋白质或RNA分子的表达 特定基因表达（RNAi）。各种不同的启动子 序列使研究人员能够将转基因的表达限制为 特定的组织和开发过程中的特定时间。一些 启动子直接异常高水平的表达。这 其他启动子的表达可以以开/关的方式调节 通过用直接的特定药物处理转基因动物 启动子的活动。编码序列可以编码 蛋白质或RNAi分子是野生型或突变体。 突变蛋白包括那些异常活跃的蛋白质以及 那些充当正常的负面抑制器 内源性蛋白活性。 标准转基因技术涉及 转基因DMA序列到单细胞胚胎的原始序列中。 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。此插入是随机的，是个性的 胚胎将包含在不同基因座的DNA插入 基因组。通常，每个胚胎基因组中只有一个位置 插入。插入的位置极大地影响了 转基因的表达。而发起人和其他监管 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 导致一定程度的随机性在表达模式中 标准转基因。因此，一旦建立了一条线， 必须评估转基因的表达。 显微注射DNA后，允许单细胞胚胎 一夜之间在体外发展。典型的注射会导致50-80％ 注射的胚胎发展到两个细胞阶段。这些 胚胎植入寄养女性，怀孕是 允许任期。幼崽出生的幼虫被指定为fo 一代。单独的小鼠幼崽是基因分型来识别的 保留了注射的转基因。这些fo动物是育种的 当成熟以识别那些传递转基因的人 一代（F1）。演示转基因传输到 F1生成和转基因表达水平的评估 模式是建立新的最终步骤 转基因线。