Mosaic Analysis with Double Markers in Mice

小鼠双标记马赛克分析

• 批准号: 8006387
• 负责人: LIQUN LUO
• 金额: $ 36.32万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8006387
• 关键词: Animal Model; Animals; Biological Process; Brain; Bypass; Candidate Disease Gene; Cells; Chromosomes; Chromosomes, Human, Pair 6; Data; Development; Gene Transfer Techniques; Genes; Genetic; Genetic Recombination; Genotype; Grant; Health; Human; Individual; Introns; Knock-in Mouse; Knock-out; Label; Laboratories; Libraries; Location; Mediating; Mus; Mutate; N-Methyl-D-Aspartate Receptors; Names; Neurologic; Neurons; Organism; Pathogenesis; Pattern; Population; Proteins; Public Health; Role; Shapes; Site; Smith Magenis syndrome; Somatic Cell; Somatic Mutation; Staging; Synaptic Transmission; System; Techniques; Tetanus Helper Peptide; Time; Tissues; VAMP-2; autism spectrum disorder; base; cell type; embryonic stem cell; experience; gene function; genetic manipulation; human disease; in vivo; interest; lissencephaly; mutant; nervous system disorder; neurodevelopment; neuron development

DESCRIPTION (provided by applicant): Genetic mosaics, in which somatic cells of different genotypes reside in the same animal, have been widely used to study biological processes in multicellular organisms. By knocking out a candidate gene of interest in a defined population of cells at a desirable time, one can study gene function in biological processes of interest while bypassing possible requirements for the gene in other tissues or at earlier developmental stages. One can also analyze the cell autonomy of gene function if the candidate gene is removed only in small populations of defined cell types. In addition, mosaic analysis can be used to create animal models for human diseases that result from somatic mutations. We have recently developed a genetic mosaic system in mice termed "MADM" (for Mosaic Analysis with Double Markers), which allows simultaneous in vivo labeling and genetic manipulation of defined neuronal populations, down to the level of single isolated neurons. We have established MADM at the ROSA26 locus of mouse chromosome 6 to show that 1) inter-chromosomal recombination can occur efficiently the Cre-loxP system; 2) MADM can be used to create conditional knockouts in small populations of labeled cells to study gene function; 3) MADM can be used to investigate the relationship between neuronal lineage and wiring patterns. We have also preliminary data that MADM can be expanded to other chromosomes using targeted knockin and random ES cell transgenesis approaches. We now propose to expand the MADM system to all mouse chromosomes so that one can perform MADM-based mosaic analysis for vast majority of genes in the mouse. We also propose to use the features afforded by MADM to investigate the role of neuronal activity in morphological maturation of individual neurons and in circuit development. Lastly, we will apply MADM to study several genes implicated in human neurological diseases, including lissencephaly and autism- spectrum disorders. PUBLIC HEALTH RELEVANCE: We propose to expand a technique we developed that allows one to disrupt a gene in small population of well-defined cells in the mouse. Utilizing this technique, we will study how experience in the form of neuronal activity shapes neuronal development and brain wiring, and why certain genes, when mutated in human, cause devastating neurological problems. These studies will have direct implications to our understanding of the pathogenesis of lissencephaly and Smith-Magenis Syndrome.

描述（由申请人提供）：遗传镶嵌物，其中不同基因型的体细胞位于同一动物中，已广泛用于研究多细胞生物的生物学过程。 通过在理想的时间中淘汰定义的细胞群中感兴趣的候选基因，人们可以在感兴趣的生物学过程中研究基因功能，同时绕过其他组织中对基因的可能要求或在早期发育阶段。 如果仅在定义的细胞类型的小种群中除去候选基因，则还可以分析基因功能的细胞自主权。 此外，镶嵌分析可用于创建由躯体突变导致的人类疾病的动物模型。 我们最近在称为“ MADM”的小鼠中开发了一个遗传镶嵌系统（用于带有双标记的镶嵌分析），该系统允许同时在体内标记和对定义神经元群体的遗传操纵，直至单个孤立神经元的水平。 我们已经在小鼠染色体6的Rosa26基因座建立了MADM，以表明1）染色体间重组可以有效地发生Cre-loxp系统； 2）MADM可用于在少数标记的细胞种群中创建条件敲除以研究基因功能； 3）MADM可用于研究神经元谱系和接线模式之间的关系。 我们还拥有初步数据，即可以使用靶向的敲击蛋白和随机ES细胞转基因方法将MADM扩展到其他染色体。 现在，我们建议将MADM系统扩展到所有小鼠染色体，以便可以对小鼠中绝大多数基因进行基于MADM的镶嵌分析。 我们还建议使用MADM提供的特征来研究神经元活动在单个神经元形态成熟和电路发育中的作用。 最后，我们将使用MADM来研究与人类神经系统疾病有关的几种基因，包括脑脑和自闭症谱系疾病。 公共卫生相关性：我们建议扩展一种我们开发的技术，该技术可以破坏小鼠中少量定义明确的细胞中的基因。 利用这项技术，我们将研究神经元活动形式的经验如何塑造神经元的发育和脑接线，以及为什么在人类中突变的某些基因会导致毁灭性神经系统问题。 这些研究将对我们对Lissencephaly和Smith-Magenis综合征的发病机理的理解有直接的影响。