Mosaic Analysis with Double Markers in Mice

小鼠双标记马赛克分析

• 批准号: 6858492
• 负责人: LIQUN LUO
• 金额: $ 36.57万
• 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 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6858492
• 关键词: gene expression; gene mutation; genetic markers; genetic recombination; genetically modified animals; genotype; histology; laboratory mouse; laboratory rat; neurons; polymerase chain reaction; southern blotting; technology /technique development; tissue mosaicism

DESCRIPTION (provided by applicant): We propose to develop a genetic mosaic system in mice that allows simultaneous labeling and genetic manipulation of defined neuronal populations, down to the level of single isolated neurons in vivo. This system, which we have named "MADM" (for Mosaic Analysis with Double Markers), utilizes two hybrid marker genes knocked-in at identical locations on homologous chromosomes. Each marker gene is interrupted by a loxP-containing intron and neither expresses a functional protein. Only upon Cre-mediated recombination between the two loxP sites on the homologous chromosomes are functional marker genes restored. Depending on the cell-cycle stage at which recombination takes place and the segregation pattern of the chromosomes after recombination, daughter cells are labeled with one or both markers. We have preliminary results indicating that MADM can be used to generate, with high efficiency, inter-chromosomal exchanges in both postmitotic neurons and in dividing neural precursors. By further developing this method and its variations we will be able to label defined neuronal populations and single neurons with genetically encoded markers, in live or fixed brains. It will also be possible to create genetic mosaics such that cells expressing the first functional marker are homozygous mutant for a gene of interest, whereas cells expressing the second functional marker are homozygous wild type, while the rest of the animal is heterozygous. MADM will allow investigation of the relationship between cell lineage and neural circuits during development, tracing of neural circuits in the adult nervous system, and conditional knock-out of candidate genes of interest as well as overexpression of transgenes in single isolated neurons. This method can also be used to create mouse models for human diseases such as loss of heterozygosity in cancer and neurological diseases.

描述（由申请人提供）：我们建议在小鼠中开发一种遗传嵌合系统，该系统允许对确定的神经元群体进行同时标记和遗传操作，直至体内单个分离神经元的水平。我们将这个系统命名为“MADM”（双标记镶嵌分析），它利用在同源染色体上相同位置敲入的两个杂交标记基因。每个标记基因都被含有 loxP 的内含子打断，并且都不表达功能蛋白。只有在同源染色体上两个 loxP 位点之间发生 Cre 介导的重组后，功能标记基因才会恢复。根据重组发生的细胞周期阶段以及重组后染色体的分离模式，子细胞被一种或两种标记物标记。我们的初步结果表明，MADM 可用于在有丝分裂后神经元和分裂神经前体中高效地产生染色体间交换。通过进一步开发这种方法及其变体，我们将能够在活体或固定大脑中用基因编码标记标记确定的神经元群体和单个神经元。还可以创建遗传嵌合体，使得表达第一功能标记的细胞是感兴趣基因的纯合突变体，而表达第二功能标记的细胞是纯合野生型，而动物的其余部分是杂合的。 MADM 将允许研究发育过程中细胞谱系和神经回路之间的关系，追踪成体神经系统中的神经回路，有条件地敲除感兴趣的候选基因以及单个分离神经元中转基因的过度表达。该方法还可用于创建人类疾病的小鼠模型，例如癌症和神经系统疾病的杂合性缺失。