A more efficient Big Blue protocol improves transgene rescue and accuracy in a adduct and mutation measurement.
Transgenic mutational systems have provided researchers with an invaluable tool, allowing the measurement of both spontaneous and induced mutations. The Big Blue transgenic rodent mutagenesis system developed by Stratagene (La Jolla, CA) uses a lambda shuttle vector carrying lacI as the mutational target gene. A common criticism of the Big Blue system is that it relies on visual screening to detect mutants rather than positive selection, which is employed in more recently developed systems. The lack of positive selection, however, has provided the Big Blue system with a unique advantage, as it allows for the dynamic quantification of mutation fixation, repair, and adduct stability, since both pre-mutagenic DNA adducts and mutations can readily be quantified [Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 11391]. Improvements to the standard Big Blue assay protocol are required for the visualization of mutant plaques resulting from pre-mutagenic damage, as these can appear much lighter in color than the lightest color control mutant (CM0). This increase in detection has been achieved by the development of a protocol that now permits the effective measurement of repair and mutation fixation utilizing the Big Blue system. This new protocol has also addressed efficiency, allowing for a two-fold increase in the number of plaques produced per packaging reaction and a decrease in both phage migration and plaque size, permitting a greater than three-fold increase in plating density. The implementation of this protocol will make the Big Blue assay more economical and less demanding than before, while providing researchers with an efficient means to measure both repair and mutation in this system.
Bielas
Jason H
JH
Department of Biology, York University, Toronto, Ont., Canada M3J 1P3. jbielas@yorku.ca
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D010582:Bacteriophage lambda / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D018736:DNA Adducts / Q000032:analysis*; D004252:DNA Mutational Analysis / Q000379:methods*; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D005038:Ethylnitrosourea / Q000633:toxicity; D056968:Lac Repressors; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D010948:Viral Plaque Assay
D001426:Bacterial Proteins; D018736:DNA Adducts; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D009153:Mutagens; D012097:Repressor Proteins; D005038:Ethylnitrosourea