The Lambda Select cII Mutation Detection System

Summary

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

Abstract

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Introduction

A wide range of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic Big Blue (referred to hereafter as BB) mice and the λ Select cII Mutation Detection System have been employed for mutagenicity experiments by this group and many other research groups worldwide^{1,2,3,4,5,6,7,8,}....

Protocol

1. Genomic DNA Isolation from Mouse Embryonic Fibroblasts

NOTE: Primary mouse embryonic fibroblasts are isolated from embryos derived from BB transgenic mice with C57BL/6 genetic background, according to the published protocol⁵³. The starting material for this protocol consists of 1 x 10⁶ to 1 x 10⁷ embryonic fibroblast cells treated with a test compound versus control. The harvesting and counting of these cells using standard methods are described in references^10,54,55.

1. Prepare buf....

Results

Depending on data distribution, parametric or non-parametric tests are used to determine the significance of difference in the cII mutant frequency between treatment and control groups (i.e., induced versus spontaneous mutant frequencies). Comparison of the induced cII mutant frequencies across different treatment groups is made by various (pairwise) statistical tests, as applicable. The hypergeometric test of Adams and Skopek is commonly used to compare the ove.......

Discussion

The λ Select cII assay is used for detection of mutations in the cII transgene recovered from the genomic DNA of cells derived from organs/tissues of BB rodents³. The genome of these transgenic animals contains multiple tandem copies of the chromosomally integrated λLIZ shuttle vector, which carries the cII (294 bp) and lacI (1,080 bp) transgenes, as the mutational reporter genes^1,2,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	MO Bio Laboratories, Inc.	12112-05	Bacteriological grade
BigDye Terminator v3.1 Cycle Sequencing Kit	Thermo Fisher Scientific	4337455	None
Casein Peptone	Alfa Aesar	H26557	None
Gelatine	J. T. Baker	2124-01	Powder
Glycerol	Fisher Scientific	BP 229-1 / M-13750	None
LB Agar	Fisher Scientific	BP 9724-500	None
QIAquick PCR purification kit	Qiagen	8104	50 PCR purification reactions
Sodium Acetate Trihydrate	Fisher Scientific	M-15756	None
Taq5000 DNA Polymerase	Qiagen	201207	None
Thiamine Hydrochloride	Macron Fine Chemicals	2722-57	None
Transpack Packaging Extract	Stratagene Corp., Acquired by BioReliance | Sigma-Aldrich Corp.	200223	50 packaging reactions
Tris Base	Fisher Scientific	BP 152-1 / EC 201-064-4	None
Trypton	Biosciences	RC-110	None