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

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.