Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice
Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice
复制标题
DOI：
10.1073/pnas.1232231100
复制
发表时间：
2003-05-27
影响因子：
11.1
通讯作者：
Engelward, BP
中科院分区：
文献类型：
--
作者：
Hendricks, CA;Almeida, KH;Engelward, BP
一种转基因小鼠已经被创造出来，它为揭示调节有丝分裂同源重组的遗传和环境因素提供了一个强大的工具。本文描述的荧光黄色直接重复序列(FYDR)小鼠携带两个不同拷贝的表达盒，用于截断的增强黄色荧光蛋白(EYFP)编码序列，串联排列。这些重复元件之间的同源重组可以恢复全长EYFP编码序列以产生荧光表型，所得到的荧光重组细胞可以通过流式细胞仪快速定量。对重组FYDR细胞基因组DNA的分析表明，该小鼠模型检测到基因转换，并且基于整合的重组底物的排列，不相等的姐妹染色单体交换和修复折叠的复制叉也有望重建EYFP编码序列。来自成人耳组织的原代成纤维细胞的自发重组率为每10(6)个细胞分裂1.3+/-0.1。有趣的是，在来自胚胎组织的成纤维细胞中，这一比率大约高出10倍。我们观察到，当接触丝裂霉素C时，耳部成纤维细胞培养中重组细胞的频率大约增加了15倍，这与链间交联诱导同源重组的能力是一致的。除了研究培养的原代细胞中的重组外，还通过直接分析分解的细胞来测量皮肤中存在的重组细胞的频率。因此，FYDR小鼠模型可用于体外和体内有丝分裂同源重组的研究。
A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is; 1.3+/-0.1 per 10(6) cell divisions. Interestingly, the rate is approximate to10-fold greater in fibroblasts derived from embryonic tissue. We observe an approximate to15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.