GENE MANIPULATION CORE

基因操纵核心

基本信息

  • 批准号:
    7699756
  • 负责人:
  • 金额:
    $ 19.2万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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

项目成果

期刊论文数量(0)
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会议论文数量(0)
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Christopher A. Walsh其他文献

Unveiling causal regulatory mechanisms through cell-state parallax
通过细胞状态视差揭示因果调节机制
  • DOI:
    10.1101/2023.03.02.530529
  • 发表时间:
    2023
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Alexander P. Wu;Rohit Singh;Christopher A. Walsh;Bonnie Berger
  • 通讯作者:
    Bonnie Berger
Spatial transcriptomics reveals human cortical layer and area specification
空间转录组学揭示人类皮质层和区域的特化
  • DOI:
    10.1038/s41586-025-09010-1
  • 发表时间:
    2025-05-14
  • 期刊:
  • 影响因子:
    48.500
  • 作者:
    Xuyu Qian;Kyle Coleman;Shunzhou Jiang;Andrea J. Kriz;Jack H. Marciano;Chunyu Luo;Chunhui Cai;Monica Devi Manam;Emre Caglayan;Abbe Lai;David Exposito-Alonso;Aoi Otani;Urmi Ghosh;Diane D. Shao;Rebecca E. Andersen;Jennifer E. Neil;Robert Johnson;Alexandra LeFevre;Jonathan L. Hecht;Nicola Micali;Nenad Sestan;Pasko Rakic;Michael B. Miller;Liang Sun;Carsen Stringer;Mingyao Li;Christopher A. Walsh
  • 通讯作者:
    Christopher A. Walsh
Mechanisms of cerebral cortical patterning in mice and humans
小鼠和人类大脑皮质模式形成的机制
  • DOI:
    10.1038/nn752
  • 发表时间:
    2001-10-29
  • 期刊:
  • 影响因子:
    20.000
  • 作者:
    Edwin S. Monuki;Christopher A. Walsh
  • 通讯作者:
    Christopher A. Walsh
Bi-allelic variants in emINTS11/em are associated with a complex neurological disorder
emINTS11 中的双等位基因变异与复杂的神经系统疾病有关。
  • DOI:
    10.1016/j.ajhg.2023.03.012
  • 发表时间:
    2023-05-04
  • 期刊:
  • 影响因子:
    8.100
  • 作者:
    Burak Tepe;Erica L. Macke;Marcello Niceta;Monika Weisz Hubshman;Oguz Kanca;Laura Schultz-Rogers;Yuri A. Zarate;G. Bradley Schaefer;Jorge Luis Granadillo De Luque;Daniel J. Wegner;Benjamin Cogne;Brigitte Gilbert-Dussardier;Xavier Le Guillou;Eric J. Wagner;Lynn S. Pais;Jennifer E. Neil;Ganeshwaran H. Mochida;Christopher A. Walsh;Nurit Magal;Valerie Drasinover;Hugo J. Bellen
  • 通讯作者:
    Hugo J. Bellen
APP gene copy number changes reflect exogenous contamination
APP 基因拷贝数变化反映了外源性污染
  • DOI:
    10.1038/s41586-020-2522-3
  • 发表时间:
    2020-08-19
  • 期刊:
  • 影响因子:
    48.500
  • 作者:
    Junho Kim;Boxun Zhao;August Yue Huang;Michael B. Miller;Michael A. Lodato;Christopher A. Walsh;Eunjung Alice Lee
  • 通讯作者:
    Eunjung Alice Lee

Christopher A. Walsh的其他文献

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{{ truncateString('Christopher A. Walsh', 18)}}的其他基金

Somatic mutations in epilepsy: whole genome sequence analysis of single neurons
癫痫的体细胞突变:单个神经元的全基因组序列分析
  • 批准号:
    8333652
  • 财政年份:
    2012
  • 资助金额:
    $ 19.2万
  • 项目类别:
Somatic mutations in epilepsy: whole genome sequence analysis of single neurons
癫痫的体细胞突变:单个神经元的全基因组序列分析
  • 批准号:
    8585129
  • 财政年份:
    2012
  • 资助金额:
    $ 19.2万
  • 项目类别:
Somatic mutations in epilepsy: whole genome sequence analysis of single neurons
癫痫的体细胞突变:单个神经元的全基因组序列分析
  • 批准号:
    8451280
  • 财政年份:
    2012
  • 资助金额:
    $ 19.2万
  • 项目类别:
Human autism genetics and activity dependent gene activation
人类自闭症遗传学和活动依赖性基因激活
  • 批准号:
    7854091
  • 财政年份:
    2009
  • 资助金额:
    $ 19.2万
  • 项目类别:
Human autism genetics and activity dependent gene activation
人类自闭症遗传学和活动依赖性基因激活
  • 批准号:
    7941723
  • 财政年份:
    2009
  • 资助金额:
    $ 19.2万
  • 项目类别:
Genetic Analysis of Microcephaly in Tunisian Population
突尼斯人群小头畸形的遗传分析
  • 批准号:
    7429860
  • 财政年份:
    2008
  • 资助金额:
    $ 19.2万
  • 项目类别:
Autism genetics: homozygosity mapping and functional validation
自闭症遗传学:纯合性作图和功能验证
  • 批准号:
    8531350
  • 财政年份:
    2007
  • 资助金额:
    $ 19.2万
  • 项目类别:
Finding Autism Genes by Genomic Copy Number Analysis
通过基因组拷贝数分析寻找自闭症基因
  • 批准号:
    7872965
  • 财政年份:
    2007
  • 资助金额:
    $ 19.2万
  • 项目类别:
INVESTIGATION OF THE CLINICAL FEATURES OF PERIVENTRICULAR NODULAR HETEROTOPIA
脑室周围结节性异位的临床特征探讨
  • 批准号:
    7606921
  • 财政年份:
    2007
  • 资助金额:
    $ 19.2万
  • 项目类别:
Finding Autism Genes by Genomic Copy Number Analysis
通过基因组拷贝数分析寻找自闭症基因
  • 批准号:
    7631226
  • 财政年份:
    2007
  • 资助金额:
    $ 19.2万
  • 项目类别:

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腺苷 A1 受体中变构驱动的 G 蛋白选择性
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