Visualizing loss of heterozygosity in living mouse cells and tissues
Visualizing loss of heterozygosity in living mouse cells and tissues
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DOI：
10.1016/j.mrfmmm.2008.07.009
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发表时间：
2008-10-14
影响因子：
2.3
通讯作者：
Stringer, James R.
中科院分区：
文献类型：
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作者：
Fischer, Jared M.;Stringer, James R.
体细胞杂合性缺失 (LOH) 可能会导致癌症的发生，但对于身体正常细胞中 LOH 发生的频率知之甚少。为了原位检测 LOH，我们研究了小鼠 shYFP 胚胎干 (ES) 细胞和源自这些 ES 细胞的肠上皮细胞。 shYFP ES 细胞在 ROSA26 基因座上是杂合的。该基因座的一个拷贝携带编码黄色荧光蛋白 (YFP) 的基因，而另一个拷贝则携带 shRNA 基因，该基因产生短发夹 RNA (shRNA) 分子，导致 YFP mRNA 降解。 shYFP 群体中几乎所有细胞都发出微弱的荧光，但明亮的荧光细胞以大约 10(-5) 个明亮细胞/代的速率出现。明亮细胞缺乏编码 shRNA 的基因，并含有两个 YFP 基因拷贝。将这些结果与先前缺乏干扰 shRNA 的 ES 细胞中 LOH 的数据进行比较表明，shYFP 细胞中的 LOH 不受 shRNA 存在的影响。在通过向囊胚注射 shYFP 细胞而制成的嵌合小鼠的肠绒毛中也发现了明亮的细胞。这些数据表明，这种方法可以检测 LOH，并表明它将允许在由 shYFP 细胞制成的转基因小鼠中检测多种组织和细胞类型中的 LOH。 (C) 2008 Elsevier B.V. 保留所有权利。
Loss of heterozygosity (LOH) in somatic cells can contribute to the genesis of cancer, but little is known about the frequency with which LOH occurs in normal cells of the body. To detect LOH in situ, we studied mouse shYFP embryonic stem (ES) cells and cells of the intestinal epithelia derived from these ES cells. shYFP ES cells are heterozygous at the ROSA26 locus. one copy of the locus carries a gene encoding a yellow fluorescent protein (YFP), while the other copy harbors an shRNA gene that produces a short hairpin RNA (shRNA) molecule that causes degradation of YFP mRNA. Nearly all cells in shYFP populations were faintly fluorescent, but brightly fluorescent cells arose at a rate of approximately 10(-5) bright cells/generation. Bright cells lacked the gene encoding the shRNA and contained two copies of the YFP gene. Comparison of these results to previous data on LOH in ES cells that lacked interfering shRNA showed that LOH in shYFP cells was not influenced by the presence of the shRNA. Bright cells were also seen in intestinal villi of chimeric mice made by injecting blastocysts with shYFP cells. These data demonstrate that this approach can detect LOH and suggest that it will allow detection of LOH in a broad array of tissues and cell types in transgenic mice made from shYFP cells. (C) 2008 Elsevier B.V. All rights reserved.