( Rat and mouse embryonic cell lines ar...)Big Blue Rodent Cell Cultures for Mutagenesis Studies

Rat and mouse embryonic cell lines are useful for in vitro mutagenesis studies

Denise L. Wyborski
Stratagene Cloning Systems, Inc.

The Big Blue rat cell line and the Big Blue transgenic mouse embryonic cell line are useful for in vitro mutagenesis studies. These cell lines are part of the Big Blue transgenic rodent mutagenesis assay system,* a system for detecting mutations from several known mutagens in a variety of tissues from transgenic mice and rats.^1-4

figure 1

The Big Blue rat cell line is derived from a Rat2 embryonic fibroblast cell line transfected with the Big Blue lambda shuttle vector and the pSV2Neo plasmid, which provides an antibiotic-selection marker.^5-7 The Big Blue lambda shuttle vector (figure 1) is 45 kb in size and contains the lacI and alphalacZ genes. Each cell contains 50 to 70 copies of the lambda shuttle vector integrated into the genome at two sites. The Big Blue rat cell line is resistant to the antibiotic G418 and is a thymidine kinase mutant; the chromosome number is polyploid. The spontaneous mutant frequency of this cell line is approximately 3.7 x 10⁵.

The Big Blue transgenic mouse embryonic cell line is derived from embryonic cells of transgenic mice containing the Big Blue lambda shuttle vector. The cell line was immortalized by chemical transformation and differentiation and has survived more than 50 generations. Each cell contains at least 40 copies of the Big Blue lambda shuttle vector integrated into the genome of the cell at the identical location as in the genome of the transgenic mice. This cell line exhibits a spontaneous mutant frequency of approximately 9.28 x 10⁵.

The Big Blue cell lines use the lacI gene of the Big Blue lambda shuttle vector as the target for mutagenesis. After the cells are treated with a test compound, in vitro packaging rapidly and efficiently recovers the lambda shuttle vector from the genomic DNA of the cell. These cell lines exhibit rescue efficiencies between 5x10⁴ and 1x10⁵ plaque-forming units per g of genomic DNA. Bacterial detection systems screen for mutations in the lacI target gene. The target gene can then be recovered from mutant plaques for sequence analysis.

In addition to short-term in vitro mutagenesis studies, these cell lines are useful for various toxicological applications, studies of the effects of oncogenes and tumor suppressor genes on mutation frequencies and studies of DNA repair.

REFERENCES

1. Kohler, S.W., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 78597962.

2. Dycaico, M.J., et al. (1994) Mutat. Res. 307: 461478.

3. Rogers, B.J., et al. (1995) Mutat. Res. 327: 5766.

4. Dycaico, M.J., et al. (1996) Carcinogenesis 17: 23472356.

5. Wyborski, D.L., Malkhosyan, S., Moores, J.C., Dycaico, M.J., and Short, J.M. (1994) Strategies 7: 5556.

6. Wyborski, D.L., et al. (1995) Mutat. Res. 334: 161165.

7. Manjanatha, M.G., et al. (1996) Environ. Mol. Mutagen. 27: 44