Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells
Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells
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DOI：
10.1111/j.1365-2443.2006.00944.x
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发表时间：
2006-04-01
期刊：
影响因子：
2.1
通讯作者：
Koyama, H
中科院分区：
文献类型：
--
作者：
So, S;Nomura, Y;Koyama, H
同源重组基因打靶是研究基因功能的有力工具，但在人类细胞系中的打靶效率太低，难以产生基因敲除突变体。导致布卢姆综合征(BLM)的基因缺失的几个细胞系已经显示出更高的靶向效率。因此，我们推测，通过RNA干扰瞬时抑制BLM的表达将增强基因靶向性。为了验证这一点，我们构建了一个基因校正分析系统，利用中断的次黄嘌呤磷酸核糖基转移酶(HPRT)基因座在人HT1080细胞系中测量基因打靶频率，并检测针对BLM的小干扰RNA(SiRNA)对基因打靶的影响。当用BLM siRNA处理HPRT缺失细胞时，siRNA和基因校正载体共转染后，基因打靶频率提高了3倍，而随机整合频率受到轻微影响。值得注意的是，在来自HPRT缺失细胞的BLM杂合(+/-)细胞中，BLM siRNA处理给出了五倍以上的靶向频率，即使是用于BLM+/+细胞的BLM siRNA数量的十分之一。此外，在人类前B细胞系NALM-6中，siRNA处理使HPRT和腺嘌呤磷酸核糖基转移酶(APRT)基因靶向性分别提高了6.3倍和5.8倍。这些结果表明，通过siRNA瞬时抑制BLM的表达可以刺激人类细胞的基因打靶，从而促进人类细胞系基因打靶方案的进一步改进。
Gene targeting via homologous recombination is a powerful tool for studying gene function, but the targeting efficiency in human cell lines is too low for generating knockout mutants. Several cell lines null for the gene responsible for Bloom syndrome, BLM, have shown elevated targeting efficiencies. Therefore, we reasoned that gene targeting would be enhanced by transient suppression of BLM expression by RNA interference. To test this, we constructed a gene correction assay system to measure gene targeting frequencies using a disrupted hypoxanthine phosphoribosyltransferase (HPRT) locus in the human HT1080 cell line, and examined the effect of small interfering RNA (siRNA) for BLM on gene targeting. When HPRT-null cells pretreated with BLM siRNA were co-transfected with the siRNA and a gene correction vector, the gene targeting frequency was elevated three-fold, while the random integration frequency was marginally affected. Remarkably, in BLM heterozygous (+/-) cells derived from HPRT-null cells, the BLM siRNA treatment gave more than five-fold higher targeting frequencies, even with one-tenth the amount of BLM siRNA used for BLM+/+ cells. Furthermore, in the human pre-B cell line Nalm-6, the siRNA treatment enhanced gene targeting 6.3-fold and > 5.8-fold at the HPRT and adenine phosphoribosyltransferase (APRT) loci, respectively. These results indicate that transient suppression of BLM expression by siRNA stimulates gene targeting in human cells, facilitating a further improvement of gene targeting protocols for human cell lines.