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。总体目标