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.

Database for exchangeable gene trap clones: Pathway and gene ontology analysis of exchangeable gene trap clone mouse lines

“…We found that most of the 5′RACE‐failed clones trapped the rRNA gene (Nakahara et al . ); therefore, we did not analyze these flanking genomic sequences further.…”

“…; Nakahara et al . ). In these papers, the phenotypes of the following mouse lines were analyzed: Ayu21‐18 (trapped gene; Bnc2), Ayu21‐81 (Kcnk5/TASK‐2), Ayu21‐127 (Lgr4), Ayu21‐T2 (Msi2), Ayu21‐T93 (Ccdc55/NSrp70), Ayu21‐T346 (Hmbox1/Hot1), Ayu21‐W34 (Cadm1/Igsf4), and Ayu21‐B6T44 (Cd99).…”

“…Furthermore, we reported the usefulness of Mol/MSM‐1 ES cells established from MSM/Ms mice (Nakahara et al . ), and as a result, Ayu21‐MT1–Ayu21‐MT142 (94 ES cell lines) were registered in EGTC, and 26 mouse lines were established and deposited in CARD. Consequently, 599 (1162–469–94) ES cell lines and 258 (454–170–26) mouse lines are reported here for the first time.…”

“…The ES cell lines were grown as described previously (Nakahara et al 2013). For electroporation with gene trap vectors, SpeI-digested plasmid DNA was used.…”

100-Gbit/s hybrid optoelectronic packet switching technologies for data-centric future networks

“…Figure 4 shows the structure of a novel OCPTG circuit [9]. A very short optical pulse of about 10 ps is generated by an OEIC called an OCG upon the arrival of an optical packet, and then fed into a fiber loop equipped with a semiconductor optical amplifier (SOA) and a saturable absorber (SA).…”