Restoration of mismatch repair functions in human cell line Nalm-6, which has high efficiency for gene targeting.
Restoration of mismatch repair functions in human cell line Nalm-6, which has high efficiency for gene targeting.
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DOI：
10.1371/journal.pone.0061189
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Nohmi T
中科院分区：
文献类型：
--
作者：
Suzuki T;Ukai A;Honma M;Adachi N;Nohmi T
基因打靶是反向遗传学中的一种强有力的方法。然而，这种方法在大多数人类细胞系中受到了阻碍，因为靶向效率很低。NALM-6是一种人类前B期急性淋巴细胞白血病细胞系，具有极高的基因打靶效率，用于DNA修复及相关研究领域。尽管如此，该细胞系的使用仍然受到限制，部分原因是它缺乏错配修复复合体的组成部分MSH2的表达，这导致了基因组的不稳定性增加。在这里，我们报告成功地恢复了MSH2在NALM-6细胞中的表达，并证明了恢复不会影响高靶向效率。我们通过在MSH2基因外显子8下游引入对应于外显子9至16的cDNA序列来恢复表达。内源性外显子9~16在细胞系中缺失。MSH2的表达显著降低了HPRT的自发突变频率。此外，表达MSH2的细胞中的基因打靶效率与缺失MSH2的细胞中的基因打靶效率相似。事实上，我们在MSH2熟练的NALM-6细胞中产生了杂合的REV3L基因敲除和催化死亡突变体，效率为20-30%。建立的细胞系NALM-6-MSH+对人类细胞的反向遗传学有用。
Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.
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