Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1
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DOI：
10.3791/2468
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发表时间：
2011-02-01
影响因子：
1.2
通讯作者：
Ransburgh, Derek
中科院分区：
文献类型：
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作者：
Parvin, Jeffrey;Chiba, Natsuko;Ransburgh, Derek
错义突变的功能分析可能因细胞中内源性蛋白质的存在而复杂化。BRCA 1的结构-功能分析由于缺乏全长BRCA 1蛋白的稳健测定以及表达亚型BRCA 1蛋白的细胞系的固有困难而变得复杂（1，2，3，4，5）。我们开发了一种系统，其中细胞中的内源性BRCA 1蛋白通过靶向BRCA 1 mRNA的3 '-UTR的RNAi被急性耗尽，并通过共转染表达BRCA 1变体的质粒来替代。这种方法的一个优点是BRCA 1的急性沉默和同时的替换允许细胞生长，而不会随着时间的推移而发生继发性突变或适应，以补偿BRCA 1功能的丧失。这种消耗和添加回程序是在HeLa衍生的细胞系中进行的，该细胞系易于测定同源重组活性。同源重组试验基于先前发表的方法，其中重组底物整合到基因组中（图1）（6、7、8、9）。该重组底物在失活的GFP等位基因内具有稀切I-SceI限制酶位点，并且下游是第二失活的GFP等位基因。表达I-SceI的质粒的转染导致双链断裂，其可通过同源重组修复，并且如果同源重组确实修复断裂，则其产生活性GFP等位基因，其易于通过流式细胞术对GFP蛋白表达进行评分。内源性BRCA 1的缺失导致同源重组活性降低8-10倍，而野生型质粒的加回完全恢复了同源重组功能。当全长BRCA 1的特定点突变体从共转染质粒表达时，可以对特定错义突变体的效果进行评分。例如，BRCA 1（M18 T）蛋白（一种临床意义未知的变体）在这些细胞中表达，但未能恢复BRCA 1依赖性同源重组。相比之下，另一种变异体BRCA 1（I21 V）的表达完全恢复了BRCA 1依赖性同源重组功能，其意义也未知。这种测试BRCA 1错义突变功能的策略已被应用于另一种测定中心体功能的生物系统（Kais et al，unpublished observations）。总的来说，这种方法适用于分析任何基因中必须重新分析的错义突变体。
The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein(1,2,3,4,5). We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)(6,7,8,9). This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance(10), was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.