Mosaic Analysis with Double Markers in Mice

描述(申请人提供)：我们建议在小鼠身上开发一种遗传马赛克系统，允许同时标记和遗传操作定义的神经元群体，直到体内单个分离神经元的水平。这个系统，我们已经命名为“MADM”(用于双标记镶嵌分析)，利用两个杂交标记基因在同源染色体上相同的位置敲入。每个标记基因都被一个含有loxP的内含子打断，并且都不表达功能蛋白。只有在Cre介导的同源染色体上两个loxP位点之间的重组后，功能标记基因才能恢复。根据发生重组的细胞周期阶段和重组后染色体的分离模式，子代细胞被标记一个或两个标记。我们的初步结果表明，MADM可以高效地在有丝分裂后神经元中产生染色体间交换，并用于神经前体的分裂。通过进一步发展这种方法及其变体，我们将能够在活的或固定的大脑中用遗传编码标记来标记定义的神经元群体和单个神经元。也有可能创造出遗传嵌合体，使得表达第一个功能标记的细胞是目标基因的纯合子突变，而表达第二个功能标记的细胞是纯合子野生型，而动物的其余部分是杂合子。 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，追踪成人神经系统中的神经回路，以及在单个分离的神经元中有条件地敲除感兴趣的候选基因和过量表达转基因。这种方法也可以用来建立人类疾病的小鼠模型，例如癌症和神经系统疾病中的杂合性丧失。

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.