Restoration of mismatch repair functions in human cell line Nalm-6, which has high efficiency for gene targeting

PLoS One. 2013 Apr 15;8(4):e61189. doi: 10.1371/journal.pone.0061189. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line
  • Comparative Genomic Hybridization
  • DNA Mismatch Repair*
  • DNA Repair
  • DNA-Binding Proteins / genetics
  • DNA-Directed DNA Polymerase / genetics
  • Exons
  • Gene Expression
  • Gene Knockout Techniques
  • Gene Order
  • Gene Targeting*
  • Genetic Loci
  • Genome, Human
  • Humans
  • Hypoxanthine Phosphoribosyltransferase / genetics
  • MutS Homolog 2 Protein / genetics
  • RNA, Messenger / genetics

Substances

  • DNA-Binding Proteins
  • RNA, Messenger
  • Hypoxanthine Phosphoribosyltransferase
  • DNA-Directed DNA Polymerase
  • REV3L protein, human
  • MutS Homolog 2 Protein

Grant support

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT, 18201010; MEXT, 22241016 ), the Ministry of Health, Labour and Welfare, Japan (MHLW, H21-Food-General-009), the Japan Health Science Foundation (KHB1007); for cancer research from MHLW (20 designated-8) and the Food Safety Commission. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.