Gene-trap mutagenesis using Mol/MSM-1 embryonic stem cells from MSM/Ms mice
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.
- Abe K, Noguchi H, Tagawa K, Yuzuriha M, Toyoda A, Kojima T, Ezawa K, Saitou N, Hattori M, Sakaki Y, Moriwaki K, Shiroishi T (2004) Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6 J, as defined by BAC-end sequence-SNP analysis. Genome Res 14:2439–2447PubMedCrossRefGoogle Scholar
- Araki K, Imaizumi T, Sekimoto T, Yoshinobu K, Yoshimuta J, Akizuki M, Miura K, Araki M, Yamamura K (1999) Exchangeable gene-trap using the Cre/mutated lox system. Cell Mol Biol (Noisy-le-grand) 45:737–750Google Scholar
- Moriwaki K, Shiroishi T, Yonekawa H (1994) Genetics in wild mice. Japan Scientific Societies Press/Karger, Tokyo/BaselGoogle Scholar
- Nakanishi M, Tazawa H, Tsuchiya N, Sugimura T, Tanaka T, Nakagama H (2007) Mouse strain differences in inflammatory responses of colonic mucosa induced by dextran sulfate sodium cause differential susceptibility to PhIP-induced large bowel carcinogenesis. Cancer Sci 98:1157–1163PubMedCrossRefGoogle Scholar
- Taniwaki T, Haruna K, Nakamura H, Sekimoto T, Oike Y, Imaizumi T, Saito F, Muta M, Soejima Y, Utoh A, Nakagata N, Araki M, Yamamura K, Araki K (2005) Characterization of an exchangeable gene-trap using pU-17 carrying a stop codon-beta geo cassette. Dev Growth Differ 47:163–172PubMedCrossRefGoogle Scholar