Mosaic Analysis with Double Markers in Mice

描述（由申请人提供）：我们建议在小鼠中开发一种遗传镶嵌系统，该系统允许对定义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。这个系统，我们命名为“MADM”（双标记镶嵌分析），利用两个杂交标记基因敲入同源染色体上的相同位置。每个标记基因被含loxP的内含子中断，并且都不表达功能蛋白。只有在同源染色体上的两个loxP位点之间的Cre介导的重组后，功能标记基因才恢复。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，子细胞用一种或两种标记物标记。我们有初步的结果表明，MADM可用于产生，高效率，在有丝分裂后的神经元和分裂的神经前体的染色体间的交流。通过进一步开发这种方法及其变体，我们将能够在活的或固定的大脑中用遗传编码的标记物标记定义的神经元群体和单个神经元。还可以产生遗传嵌合体，使得表达第一功能性标记的细胞是目的基因的纯合突变体，而表达第二功能性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，追踪成人神经系统中的神经回路，以及感兴趣的候选基因的条件性敲除以及单个分离神经元中转基因的过表达。该方法也可用于创建人类疾病的小鼠模型，例如癌症和神经疾病中的杂合性丢失。

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.