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I-SceI 介导的双链断裂不会增加小鼠胚胎干细胞中 Dct 基因座同源重组的频率。

I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.

机构信息

Mouse functional Genetics, Institut Pasteur, Paris, France.

出版信息

PLoS One. 2012;7(6):e39895. doi: 10.1371/journal.pone.0039895. Epub 2012 Jun 26.

DOI:10.1371/journal.pone.0039895
PMID:22761925
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3383693/
Abstract

Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase (Dct) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of Dct gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI meganuclease was indeed able to introduce a DSB at the Dct locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

摘要

在天然内源性基因座上靶向诱导双链断裂 (DSB) 已被证明可提高同源重组在小鼠胚胎干细胞中替换基因的效率。多巴胺色素互变异构酶 (Dct) 的基因在黑色素细胞及其前体细胞中特异性表达。为了构建一种允许用任何感兴趣的基因替换 Dct 基因的遗传工具，我们构建了一个携带酵母 I-SceI 核酸内切酶识别位点的胚胎干细胞系，该识别位点嵌入 Dct 基因组片段中。该胚胎干细胞系用 I-SceI 表达质粒进行电穿孔，并转染了用于 DSB 修复过程的模板，该模板与 Dct 靶标具有序列同源性。I-SceI 核酸内切酶确实能够在活胚胎干细胞中 Dct 基因座处引入 DSB。然而，DSB 的诱导并没有提高基因靶向水平，这表明 I-SceI 介导 Dct 基因座同源重组的能力有限。这些数据表明，在哺乳动物细胞中，由核酸内切酶诱导的 DSB 进行的同源重组可能依赖于基因座。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/0953f4246b2c/pone.0039895.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/7c3b6b0bcaf6/pone.0039895.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/cf19a61e8695/pone.0039895.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/20c3ee4f8d2f/pone.0039895.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/22166efae031/pone.0039895.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/db7eb280542d/pone.0039895.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/0953f4246b2c/pone.0039895.g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/7c3b6b0bcaf6/pone.0039895.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/cf19a61e8695/pone.0039895.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/20c3ee4f8d2f/pone.0039895.g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/22166efae031/pone.0039895.g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/db7eb280542d/pone.0039895.g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/37cf/3383693/0953f4246b2c/pone.0039895.g006.jpg

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