Mammalian Genome

, Volume 24, Issue 5–6, pp 228–239 | Cite as

Gene-trap mutagenesis using Mol/MSM-1 embryonic stem cells from MSM/Ms mice

  • Mai Nakahara
  • Hiroki Tateyama
  • Masatake Araki
  • Naomi Nakagata
  • Ken-ichi Yamamura
  • Kimi Araki
Article

Abstract

The MSM/Ms strain is derived from the Japanese wild mouse Mus musculus molossinus and displays characteristics not observed in common laboratory strains. Functional genomic analyses using genetically engineered MSM/Ms mice will reveal novel phenotypes and gene functions/interactions. We previously reported the establishment of a germline-competent embryonic stem (ES) cell line, Mol/MSM-1, from the MSM/Ms strain. To analyze its usefulness for insertional mutagenesis, we performed gene-trapping using these cells. In the present study, we compared the gene-trap events between Mol/MSM-1 and a conventional ES cell line, KTPU8, derived from the F1 progeny of a C57BL/6 × CBA cross. We introduced a promoter-trap vector carrying the promoterless β-galactosidase/neomycin-resistance fusion gene into Mol/MSM-1 and KTPU8 cells, isolated clones, and identified the trapped genes by rapid amplification of cDNA 5′-ends (5′-RACE), inverse PCR, or plasmid rescue. Unexpectedly, the success rate of 5′-RACE in Mol/MSM trap clones was 47 %, lower than the 87 % observed in KTPU8 clones. Genomic analysis of the 5′-RACE-failed clones revealed that most had trapped ribosomal RNA gene regions. The percentage of ribosomal RNA region trap clones was 41 % in Mol/MSM-1 cells, but less than 10 % in KTPU8 cells. However, within the Mol/MSM-1 5′-RACE-successful clones, the trapping frequency of annotated genes, the chromosomal distribution of vector insertions, the frequency of integration into an intron around the start codon-containing exon, and the functional spectrum of trapped genes were comparable to those in KTPU8 cells. By selecting 5′-RACE-successful clones, it is possible to perform gene-trapping efficiently using Mol/MSM-1 ES cells and promoter-trap vectors.

Supplementary material

335_2013_9452_MOESM1_ESM.docx (13 kb)
Supplementary Material 1 (DOCX 13 kb)

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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Mai Nakahara
    • 1
  • Hiroki Tateyama
    • 1
  • Masatake Araki
    • 2
  • Naomi Nakagata
    • 3
  • Ken-ichi Yamamura
    • 1
  • Kimi Araki
    • 1
  1. 1.Division of Developmental Genetics, Institute of Resource Development and AnalysisKumamoto UniversityKumamotoJapan
  2. 2.Division of Bioinformatics, Institute of Resource Development and AnalysisKumamoto UniversityKumamotoJapan
  3. 3.Division of Reproductive Engineering, Institute of Resource Development and AnalysisKumamoto UniversityKumamotoJapan

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