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
- 来源:
- 关键词:129 MouseAdenosine A1 ReceptorAdenovirusesAdultAliquotAnimal HospitalsAnimal HusbandryAnimalsAreaArtsAtlasesBackBacteriaBacterial Artificial ChromosomesBehavioralBindingBiological AssayBiological PreservationBrainBreedingCategoriesCell SurvivalCellsChimera organismChromosomesClassificationCleaved cellCloningCloning VectorsCodeCollectionColorComplexConsultConsultationsCryopreservationCulture MediaDNADNA analysisDependovirusDetectionDevelopmentDevicesDiploidyDiseaseDominant-Negative MutationDrug resistanceES Cell LineEducational process of instructingEmbryoEmbryo TransferEndoribonucleasesEnsureEnvironmentEquipmentEventExhibitsExperimental DesignsFacility Construction Funding CategoryFemaleFigs - dietaryFosteringFreezingFundingGene ExpressionGene TargetingGene Transfer TechniquesGenerationsGenesGeneticGenetic RecombinationGenomeGenotypeGerm CellsGerm LinesGrowthGuidelinesHarvestHippocampus (Brain)HormonesHornsHousingHuman ResourcesHybridsImplantIn VitroIndividualInfectionInformation ResourcesInjection of therapeutic agentInstitutionJournalsKaryotypeKnock-in MouseKnock-outLaboratoriesLaboratory cultureLentivirus VectorLettersLiquid substanceLocationMaintenanceMammalian OviductsMedicalMental Retardation and Developmental Disabilities Research CentersMessenger RNAMethodsMicroinjectionsMouse StrainsMusMutateMutationNatural regenerationNervous system structureNeuraxisNeuronsNitrogenNumbersOperative Surgical ProceduresPartner in relationshipPatient currently pregnantPatternPediatric HospitalsPersonal SatisfactionPharmaceutical PreparationsPhenotypePhysiologicalPolymerase Chain ReactionPopulationPregnancyProceduresProcessProductionProteinsProtocols documentationQuality ControlRNARNA InterferenceRangeReagentReceptor GeneRecombinant DNARecyclingResearchResearch PersonnelResearch Project GrantsResourcesRetroviridaeRodentSafetySamplingSeriesServicesSiteSmall Interfering RNAStagingStandards of Weights and MeasuresTechnical ExpertiseTechniquesTechnologyTimeTissuesTrainingTransfectionTransgenesTransgenic AnimalsTransgenic MiceTransgenic OrganismsUterusVaginaViral VectorWorkWritingadeno-associated viral vectoranimal breedinganimal colonyanimal facilityanimal resourcebaseblastocystcostdata managementdaydesigndesign and constructiondesireeggembryo cellembryo cultureembryo tissueembryonic stem cellendoribonucleaseexperienceexpression vectorfallsgene therapygerm free conditionhomologous recombinationimplantationinsightmalemedical schoolsmembermouse genomemutantnatural Blastocyst Implantationnew technologynext generationnovelpathogenpluripotencyprogramspromoterprotein expressionpuprecombinaserepairedresearch studysmall hairpin RNAsperm cellstemsuccesstissue culturetooltransgene expressiontransmission processvectorzygote
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.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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(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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Understanding mechanisms of allostery and biased agonism at the adenosine A1 receptor
了解腺苷 A1 受体的变构和偏向激动机制
- 批准号:
nhmrc : 1084246 - 财政年份:2015
- 资助金额:
$ 19.2万 - 项目类别:
Project Grants
Analgesic actions of adenosine A1 receptor along axonal tracts in chronic pain
腺苷 A1 受体沿轴突束对慢性疼痛的镇痛作用
- 批准号:
9101984 - 财政年份:2013
- 资助金额:
$ 19.2万 - 项目类别:
Analgesic actions of adenosine A1 receptor along axonal tracts in chronic pain
腺苷 A1 受体沿轴突束对慢性疼痛的镇痛作用
- 批准号:
9294975 - 财政年份:2013
- 资助金额:
$ 19.2万 - 项目类别:














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