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Gene-trap mutagenesis using Mol/MSM-1 embryonic stem cells from MSM/Ms mice
Mai Nakahara1, Hiroki Tateyama, Masatake Araki
1Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University, Honjo 2-2-1, Kumamoto, Japan. 127y2008@st.kumamoto-u.ac.jp
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.
Insights
Gene trapping in Mol/MSM-1 embryonic stem (ES) cells, derived from Japanese wild mice, showed a lower success rate due to ribosomal RNA gene trapping. However, successful traps were comparable to conventional ES cells, enabling efficient gene function studies.
Area of Science:
- Genomics and Genetics
- Developmental Biology
- Mouse Models
Background:
- The MSM/Ms strain, derived from Japanese wild mice (Mus musculus molossinus), offers unique genetic characteristics for functional genomics.
- Germline-competent embryonic stem (ES) cells, such as Mol/MSM-1, are crucial for creating genetically engineered mouse models.
- Gene trapping is a powerful insertional mutagenesis technique to identify gene function and discover novel phenotypes.
Purpose of the Study:
- To evaluate the utility of Mol/MSM-1 ES cells for gene trapping.
- To compare gene-trap events in Mol/MSM-1 cells with those in a conventional ES cell line (KTPU8).
- To identify potential challenges and optimize gene-trapping strategies using Mol/MSM-1 cells.
Main Methods:
- Introduction of a promoter-trap vector (carrying β-galactosidase/neomycin-resistance gene) into Mol/MSM-1 and KTPU8 ES cells.
- Isolation of gene-trap clones and identification of trapped genes using 5'-rapid amplification of cDNA ends (5'-RACE), inverse PCR, and plasmid rescue.
- Genomic analysis to determine the location and frequency of vector insertions, particularly in ribosomal RNA gene regions.
Main Results:
- Mol/MSM-1 cells exhibited a lower 5'-RACE success rate (47%) compared to KTPU8 cells (87%).
- A significant proportion of failed 5'-RACE events in Mol/MSM-1 cells were attributed to trapping of ribosomal RNA gene regions (41%).
- In 5'-RACE-successful Mol/MSM-1 clones, gene-trapping efficiency, insertion patterns, and functional gene spectrum were comparable to KTPU8 cells.
Conclusions:
- Despite challenges with ribosomal RNA gene trapping, Mol/MSM-1 ES cells are suitable for efficient gene trapping when 5'-RACE-successful clones are selected.
- This study validates the use of Mol/MSM-1 ES cells for insertional mutagenesis and functional genomic studies in mice.
- The findings provide insights into optimizing gene-trapping protocols for unique mouse genetic backgrounds.
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