Mosaic Analysis with Double Markers in Mice

小鼠双标记马赛克分析

• 批准号: 7769562
• 负责人: LIQUN LUO
• 金额: $ 36.52万
• 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/7769562
• 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; 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; public health relevance

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）：遗传嵌合体，其中不同基因型的体细胞驻留在同一动物中，已被广泛用于研究多细胞生物中的生物过程。 通过在所需的时间敲除特定细胞群中的候选目标基因，人们可以研究目标生物过程中的基因功能，同时绕过其他组织或早期发育阶段对该基因的可能需求。 如果仅在一小群特定细胞类型中去除候选基因，则还可以分析基因功能的细胞自主性。 此外，镶嵌分析还可用于创建由体细胞突变引起的人类疾病的动物模型。 我们最近在小鼠中开发了一种称为“MADM”（双标记马赛克分析）的遗传镶嵌系统，该系统允许同时对确定的神经元群体进行体内标记和遗传操作，直至单个分离神经元的水平。 我们在小鼠6号染色体ROSA26位点建立了MADM，表明1）Cre-loxP系统可以有效地发生染色体间重组； 2) MADM 可用于在小群标记细胞中进行条件敲除，以研究基因功能； 3）MADM可用于研究神经元谱系和布线模式之间的关系。 我们还有初步数据表明，可以使用靶向敲入和随机 ES 细胞转基因方法将 MADM 扩展到其他染色体。 我们现在建议将 MADM 系统扩展到所有小鼠染色体，以便可以对小鼠的绝大多数基因进行基于 MADM 的镶嵌分析。 我们还建议使用 MADM 提供的功能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，我们将应用 MADM 来研究与人类神经系统疾病有关的几种基因，包括无脑畸形和自闭症谱系障碍。 公共健康相关性：我们建议扩展我们开发的一项技术，该技术允许破坏小鼠小群明确细胞中的基因。 利用这项技术，我们将研究神经元活动形式的经验如何影响神经元发育和大脑接线，以及为什么某些基因在人类体内发生突变时会导致毁灭性的神经系统问题。 这些研究将对我们了解无脑畸形和史密斯-马吉尼斯综合征的发病机制产生直接影响。