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A Positive Selection Assay for Mutation Analysis in Big Blue,,,Animals


Cost-effective and easy-to-use l Select-cII mutation assay kit

A Positive Selection Assay for Mutation Analysis in Big Blue Animals

Mark J. Dycaico Gabriela M. Tobal
Stratagene

Stratagene now offers an alternative way to perform mutational analysis in the Big Blue transgenic rodent mutagenesis assay system that selects for mutations in the cII gene of coliphage lambda. The l Select-cII mutation assay kit uses an hfl E. coli strain and selective temperature conditions to provide an environment where only lambda cII mutants form plaques. This assay not only provides a less expensive means for studying mutations in Big Blue rodents* but also complements the original Big Blue (lacI) assay by focusing on a second genetic region for studying tissue-specific mutant frequencies and mutational spectra.

figure 1

Stratagenes Big Blue transgenic rodent mutagenesis assay system has been used extensively for studying mutations sustained in vivo in mammals.1,2 Big Blue transgenic rodents harbor the Big Blue lLIZ shuttle vector (figure 1), which can easily be rescued from the genome for mutation analysis in an E. coli host. In the established version of the Big Blue assay, the lacI gene from E. coli, located within the lLIZ shuttle vector, functions as a mutational test sequence. Rescued shuttle vector phage containing lacI mutations are scored as blue plaques against a background of colorless wild-type plaques.3

In addition to providing the lacI plaque color-screening assay, Stratagene is now offering an alternative method of screening for mutations in Big Blue rodents, the l Select-cII mutation assay kit. In this positive selection assay, the region encompassing the cII gene of coliphage lambda functions as the mutational test sequence.4 The cII gene encodes a protein that activates transcriptional promoters in lambda that are essential for lysogenization. Mutations in the cII region that lower the levels of cII protein result in a decreased ability of lambda to lysogenize. When grown under conditions that favor lysogeny, lambda prophages carrying such mutations (lcII ) survive only by entering the lytic pathway of development, forming plaques. Prophages that are wild type for the cII region (lcII +) integrate into the host genome and become part of the developing bacterial lawn.

figure 2

The method of performing the l Select-cII mutation assay is outlined (figure 2). After rescuing the lLIZ shuttle vector from the rodent genomic DNA, the packaged virions are used to infect the E. coli host strain G1250 (a specialized hfl version of Stratagenes XL1-Blue MRA cells). The infected host cells are then plated and grown at 24C for 48 hours, conditions that are selective for lcII mutants (figure 2, path A). In addition, a dilution of infected G1250 cells is plated and grown under nonselective conditions (37C overnight) to determine the total number of rescued phage screened (figure 2, path B). Because the lLIZ shuttle vector contains the temperature-sensitive cI 857 mutation, both lcII + and lcII shuttle vector phages grow as plaques at 37C. Mutant frequency is determined as the number of lcII plaque-forming units (pfu) observed divided by the total pfu screened. The lcII selection scheme has been used to measure mutant frequency inductions in bladder tissue from mice treated with p-cresidine, with results comparable to lacI plaque color screening.4

The Big Blue Cyclist DNA sequencing kit can be used to rapidly sequence the lcII mutants identified with the l Select-cII assay. The cII target region is approximately 300 base pairs in length, including the cII mRNA ribosome binding site and the cII protein-activated PRE promoter.5,6 The relatively short length of the cII target region simplifies sequencing and expedites the analysis of mutational spectra.

Conclusions

The lSelect-cII mutation assay kit offers an alternative to the lacI plaque color-screening assay for mutation analysis in Big Blue transgenic rodents. Both assays feature unique advantages. The traditional plaque color-screening assay makes use of the larger, highly characterized lacI target region and benefits from the extensive database of experimental literature published over several years. By comparison, the lSelect-cII mutation assay is more cost-effective and less labor-intensive to perform due to its selective nature and small target region. This new assay can also serve to complement plaque color screening through mutation analysis of a second genetic region within the same animal. The l Select-cII mutation assay kit includes the G1250 selective host strain, enough Transpack lambda packaging extract** for 50 packaging reactions and lcII control mutants.

REFERENCES
  1. Mirsalis, J.C., Monforte, J.A., and Winegar, R.A. (1995) Annu. Rev. Pharmacol. Toxicol. 35: 145-164.

  2. Gorelick, N.J., and Mirsalis, J.C. (1996) Environ. Mol. Mutagen. 28: 434-442.

  3. Dycaico, M.J., et al. (1994) Mutat. Res. 307: 461-478.

  4. Jakubczak, J.L., et al. (1996) Proc. Natl. Acad. Sci. USA 93: 9073-9078.

  5. Schwarz, E., et al. (1978) Nature 272: 410-414.

  6. Place, N., et al. (1984) J. Mol. Biol. 180: 865-880.

  7. Rogers, B.J., et al. (1995) Mutat. Res. 327: 57-66.

  8. Young, R.R., et al. (1995) Mutat. Res. 327: 67-73.

* U.S. Patent No. 5,347,075 and patents pending. European Patent No. 289121, Japanese Patent No. 2618973
** U.S. Patent No. 5,188,957


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