Abstract
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.
Highlights
Gene targeting technology, which introduces novel DNA sequences at specific sites of the chromosome with exogenous DNA, is a powerful tool to investigate gene functions
Comparative Genome Hybridization (CGH) Array Analyses of Nalm-6 Genome To explore the cause of deficiency in mismatch repair functions in Nalm-6 cells, we first examined the proteins of MSH2 and MSH6 by the Western blotting analysis and confirmed that MSH2 was not expressed and MSH6 was poorly expressed in Nalm-6 (Fig. 1A) [20]
The transcript of the MSH6 gene was detected, no reverse transcription (RT)-polymerase chain reactions (PCR) product was detected for MSH2 gene (Fig. 1B), which were consistent with the previous report [6]
Summary
Gene targeting technology, which introduces novel DNA sequences at specific sites of the chromosome with exogenous DNA, is a powerful tool to investigate gene functions. The method is not practicable in most cases because efficiency of homologous recombination (HR) necessary for the gene targeting is much lower than that of non-homologous end joining (NHEJ) [1,2]. Recent development of zinc-finger nucleases (ZFNs) [3], which introduce double-strand breaks at unique chromosome sites and enhance local mutagenic-NHEJ and HR, appears a good candidate to overcome the limitation [4,5]. Establishment of ZFNs often requires in vivo experiments to ensure that the designed enzymes do not introduce double-strand breaks at offtarget sites. Ready-to-use ZFNs are commercially available, but numbers of genes that can be manipulated by the commercial ZFNs are still limited and made-to-order commercial ZFNs are costly
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