title: Gene-trap mutagenesis using Mol/MSM-1 embryonic stem cells from MSM/Ms mice.
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.
authors.affiliation: Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Honjo 2-2-1, Kumamoto, Japan. 127y2008@st.kumamoto-u.ac.jp
authors.forename: Mai
authors.initials: M
authors.lastname: Nakahara
authors.forename: Hiroki
authors.initials: H
authors.lastname: Tateyama
authors.forename: Masatake
authors.initials: M
authors.lastname: Araki
authors.forename: Naomi
authors.initials: N
authors.lastname: Nakagata
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yamamura
authors.forename: Kimi
authors.initials: K
authors.lastname: Araki
mesh_terms: D000818:Animals; D003433:Crosses, Genetic; D053595:Embryonic Stem Cells / Q000166:cytology*; D005260:Female; D006801:Humans; D051379:Mice / Q000235:genetics*; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D016254:Mutagenesis, Insertional