Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes
Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes
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DOI：
10.1186/1471-2199-7-36
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发表时间：
2006-10-16
影响因子：
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通讯作者：
Stringer, James R.
中科院分区：
文献类型：
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作者：
Larson, Jon S.;Yin, Moying;Stringer, James R.
背景资料：杂合性缺失（洛）导致许多癌症，但这些事件在身体正常细胞中发生的速率尚不清楚。洛缺失可在体内不同类型的细胞中检测到，如果该事件赋予明显的细胞表型的话。携带两种不同荧光蛋白基因作为同一基因座等位基因的小鼠似乎是解决这一问题的有用工具，因为洛缺失会将细胞的表型从二色性改变为单色性。此外，洛引起的有丝分裂交叉可能是可辨别的组织，因为这一事件产生了一对相邻的单色细胞，是不同的colors.Results：作为一个步骤，在评估这种方法的效用，我们得出的主要胚胎成纤维细胞群体和胚胎干细胞系的小鼠携带两种不同的荧光蛋白基因作为等位基因在染色体6位点，ROSA 26。荧光激活细胞分选（FACS）显示，每个细胞系中的绝大多数细胞以相似的水平表达两种标记蛋白，并且群体表现出与在细菌和酵母中观察到的相似的表达噪声。具有单色表型的细胞以10（-4）数量级的频率存在，并且似乎以每次有丝分裂约10（-5）个变异细胞的速率产生。45个稳定单色ES细胞克隆中的45个表现出在ROSA 26基因座处的预期等位基因的丢失。这些克隆中有一半以上在ROSA 26和着丝粒之间的基因座处保留杂合性。其他克隆在着丝粒附近出现洛缺失，但在6号染色体上为二体。结论：利用等位基因荧光标记，可以用流式细胞仪检测ROSA 26位点的洛缺失。该位点的洛通常不伴有着丝粒附近的洛，表明有丝分裂重组是ROSA 26洛的主要原因。二色性小鼠胚胎细胞为研究遗传/核型稳定性和影响等位基因表达的因素提供了一种新的系统。类似的方法将允许在组织中研究这些现象。
Background: Loss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors.Results: As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10(-4) and appeared to be produced at a rate of approximately 10(-5) variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6.Conclusion: Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.