Adachi Noritaka, So Sairei, Iiizumi Susumu, Nomura Yuji, Murai Kyoko, Yamakawa Chie, Miyagawa Kiyoshi, Koyama Hideki
Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Japan.
DNA Cell Biol. 2006 Jan;25(1):19-24. doi: 10.1089/dna.2006.25.19.
Gene targeting provides a powerful means for analyzing gene function, as exemplified by knockout mouse studies and recent work with the highly recombinogenic chicken DT40 B-lymphocyte line. In human cultured cells, however, the low frequency of gene targeting is a serious barrier to efficiently generate knockout clones. Moreover, commonly used human cell lines are karyotypically abnormal or unstable. Here, we show using promoterless targeting constructs that Nalm-6, a human pre-B ALL cell line, is highly proficient for gene targeting by homologous recombination. Indeed, the efficiency of TP53 gene targeting in Nalm-6 appears nearly two orders of magnitude higher than that in HCT116, a colon cancer cell line popularly used for gene targeting. Expression analysis revealed a lack of MSH2 expression in this cell line. As Nalm-6 has a stable neardiploid karyotype with normal p53 status, our results underscore the usefulness of Nalm-6 for gene knockout studies in humans.
基因打靶为分析基因功能提供了一种强大的手段，敲除小鼠研究以及最近利用具有高度重组能力的鸡DT40 B淋巴细胞系所开展的工作就是例证。然而，在人类培养细胞中，基因打靶的低频率是高效产生敲除克隆的一个严重障碍。此外，常用的人类细胞系在核型上是异常的或不稳定的。在此，我们使用无启动子打靶构建体表明，人前B急性淋巴细胞白血病细胞系Nalm-6通过同源重组进行基因打靶的效率很高。事实上，Nalm-6中TP53基因打靶的效率似乎比广泛用于基因打靶的结肠癌细胞系HCT116高近两个数量级。表达分析显示该细胞系中缺乏MSH2表达。由于Nalm-6具有稳定的近二倍体核型且p53状态正常，我们的结果强调了Nalm-6在人类基因敲除研究中的实用性。
