Brief expression of a GFPcre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions
Brief expression of a GFPcre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions
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DOI：
10.1093/nar/25.16.3326
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发表时间：
1997-08-15
影响因子：
14.9
通讯作者：
Sauer, B
中科院分区：
文献类型：
--
作者：
Gagneten, S;Le, YZ;Sauer, B
噬菌体P1的Cre DNA重组酶已成为通过同源重组进行基因修饰的胚胎干（ES）细胞精确基因组操作的有用工具。通过构建Cre与Aequorea victoria的增强型绿色荧光蛋白（GFPS65T）之间的功能融合，我们重新设计了ore基因，使其能够快速识别活的Cre（+）细胞。GFPcre融合基因产物在没有任何外源核定位信号的情况下快速靶向细胞核，并且在小鼠3T3衍生细胞系和小鼠ES细胞中催化了高效的DNA重组。瞬时gfpcree转染的ES细胞的荧光激活细胞分选（FAGS）不仅允许在DNA转染后快速有效地分离Cre（+）细胞，而且还证明了Cre表达的爆发足以使细胞从基因组中“弹出”Cre介导的loxp标记DNA。因此，GFPcre可以快速鉴定通过Cre介导的重组从基因组中去除loxp侧DNA序列的活细胞，而不依赖于靶位点标记基因的重组激活或失活。此外，GFPcre融合基因将被证明可用于追踪转基因动物中组织特异性Cre表达，从而促进条件基因敲除小鼠的产生和分析。
The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the ore gene to allow ready identification of living Cre(+) cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T), The GFPcre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal, Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells, Fluorescence-activated cell sorting (FAGS) of transiently GFPcre-transfected ES cells not only allowed rapid and efficient isolation of Cre(+) cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP-tagged DNA from the genome. Thus, GFPcre allows rapid identification of living cells in which loxP-flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus, In addition, the GFPcre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.