Intralaboratory optimization and standardization of mutant screening conditions used for a lambda/lacI transgenic mouse mutagenesis assay (I).
A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection. The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal. Non-mutant lacI phage plaques remain colorless. Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility. In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)). These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.
Rogers
B J
BJ
Stratagene, La Jolla, CA 92037.
Provost
G S
GS
Young
R R
RR
Putman
D L
DL
Short
J M
JM
D000362:Agar; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D010582:Bacteriophage lambda / Q000187:drug effects* / Q000235:genetics; D002863:Chromogenic Compounds; D004274:DNA, Recombinant / Q000235:genetics / Q000302:isolation & purification; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005260:Female; D005697:Galactosides; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter / Q000187:drug effects*; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000187:drug effects* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D056968:Lac Repressors; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009152:Mutagenicity Tests / Q000379:methods / Q000592:standards*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D015203:Reproducibility of Results; D010948:Viral Plaque Assay / Q000295:instrumentation; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D005697:Galactosides; D007211:Indoles; D056968:Lac Repressors; C539064:LacI protein, E coli; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D000362:Agar; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside