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.
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.
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 worldwide1,2,3,4,5,6,7,8,9. For the past 16 years, we have investigated the mutagenic effects of various chemical and/or physical agents using these transgenic animals or their corresponding embryonic fibroblast cell cultures treated with a test compound, and subsequently analyzed the phenotype and genotype of the cII transgene by the λ Select cII assay and DNA sequencing, respectively10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. The genome of these transgenic animals contains a bacteriophage λ shuttle vector (λLIZ) integrated on chromosome 4 as a multi-copy head-to-tail concatemer1,2,25. The λLIZ shuttle vector carries two mutational reporter genes, namely the lacI and cII transgenes1,2,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47. The λ Select cII assay is based on the recovery of the λLIZ shuttle vectors from the genomic DNA of cells derived from organs/tissues of transgenic animals1,2,25. The recovered λLIZ shuttle vectors are then packaged into λ phage heads capable of infecting an indicator host Escherichia coli. Subsequently, the infected bacteria are grown under selective conditions to allow for scoring and analysis of mutations in the cII transgene1,3.
Here, we describe a detailed protocol for the λ Select cII assay, which consists of isolation of genomic DNA from cells/organs of transgenic animals treated in vitro/in vivo with a test compound, retrieval of the λLIZ shuttle vectors from the genomic DNA, packaging of the vectors into infectious λ phages, infection of the host E. coli with the bacteriophages, identification of the cII-mutants under selective conditions to determine the cII mutant frequency, and DNA sequence analysis to establish the cII mutation spectrum. The protocol can be applied to transgenic mouse/rat cell cultures treated in vitro with a chemical/physical agent of interest, or tissues/organs of the corresponding animals treated in vivo with the test chemical/agent1,2,4,48,49,50,51,52. A schematic presentation of the λ Select cII assay is shown in Figure 1.
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 references10,54,55.
2. In Vitro Packaging Reaction
3. Preparing the E. coli G1250 Bacterial Culture
4. Plating the Packaged DNA Samples
5. Examining the Titer and Screening Plates to Determine the cII Mutant Frequency
6. Verification of the Putative λ cII Mutants, PCR Amplification, and DNA Sequencing
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 overall induced- and spontaneous mutation spectra57, although other tests, such as the χ2 test or Analysis of Variance (ANOVA), can also be used to compare the frequency of each specific type of mutation (e.g., transition, transversion, insertion, or deletion) between the induced- and control mutation spectra, or among various mutation spectra induced by different chemicals/agents or varying doses of the same chemical/agent.
Figure 3 is a compilation of mutant frequency data from published studies in which we have demonstrated that the extent of increase in relative cII mutant frequency in mouse embryonic fibroblasts treated with various chemicals and/or physicalagents may vary from a few- to several hundred-fold, depending on the mutagenic 'potency' of the test compound. Statistically significant fold-increases in the cII mutant frequency are shown for mouse embryonic fibroblasts treated with acrylamide12, glycidamide14, aflatoxinB1(AFB1)22, tamoxifen18, δ-aminolevulinic acid (δ-ALA) plus low dose ultraviolet light A (UVA: λ >320−400 nm)15, benzo(a)pyrene diol epoxide(B(a)PDE)19, and equilethal doses of UVA, UVB (λ =2 80−320 nm), and simulated sunlight UV (SSL) 21 (see Figure 3).
Figure 4 is a demonstration of the 'sequence-specificity' of mutations in which we have shown the induction of specific types of mutation in the cII transgene in mouseembryonicfibroblasts irradiated with UVBrelativetocontrol23.The UVB-induced mutation spectrum is characterized by significant increases in relative frequency of single- or tandem C→T transitions at pyrimidine dinucleotides.
Figure 1: Schematic presentation of the λ Select cII assay. The assay is based on the retrieval of the λLIZ shuttle vectors, which contain the cII transgene as a mutational reporter gene,from the genomic DNA of cultured cells derived from transgenic rodents treated in vitro with a test compound or tissues/organs of the corresponding animals treated in vivo with the tested chemical/agent (A and B). The rescued vectors are packaged into λ phage heads that can infect an appropriate host E. coli (C and D). The infected bacteria are then grown under selective conditions to allow for scoring and analysis of mutations in the cII transgene1,2,3,25,52 (E). Determination of the induced cII mutant frequency and establishment of the mutation spectrum by DNA sequencing are outlined in F and G. The induced- and spontaneous mutation spectra are visualized in different formats. For illustration purposes, we have highlighted a format in which the induced cII mutations are typed above the reference sequence, whereas the spontaneous mutations (control) are typed below the reference sequence (H). The height of a mutated base represents its frequency of mutations (i.e., the higher the base, the more frequently mutated). Numbers above a mutated base indicate the percentage frequency of mutations in that base. Deleted bases are underlined. Inserted bases are shown with an arrow. Numbers below the bases are reference nucleotide positions. Data are from a published study23. Please click here to view a larger version of this figure.
Figure 2: Counting plaques in titer plates. To more easily identify the plaques, plates are held next to a white light box and against a dark background with lids removed. Titer 20 plate (A) and Titer 100 plate (B). Please click here to view a larger version of this figure.
Figure 3: Mutant frequencies of the cII transgene in mouse embryonic fibroblasts treated with various chemicals and/or physical agents in comparison to controls. Data are from published studies on acrylamide12, glycidamide14, aflatoxinB1(AFB1)22, tamoxifen18, δ-aminolevulinic acid (δ-ALA) plus low dose ultraviolet light A (UVA: λ >320−400 nm)15, benzo(a)pyrenediol epoxide (B(a)PDE)19, and equilethal doses of UVA, UVB (λ = 280−320 nm), and simulated sunlight UV (SSL)21.To efficiently metabolize tamoxifen in mouse embryonic fibroblast cells, we used the S9-activation system (S9 mix) consisting of Aroclor 1,254-induced rat liver preparations and cofactor reagents22. All differences between treated- and control samples are statistically significant at p <0.05. Please click here to view a larger version of this figure.
Figure 4: Mutation spectra of the cII transgene in mouse embryonic fibroblasts irradiated with UVB relative to control. Data are from a published study23.The strand mirror counterparts of all transitions (e.g., G→A and C→T) and transversions (e.g., G→T and C→A or G→C and C→G) are combined. Ins: insertion; Del: deletion. The UVB-induced mutation spectrum is characterized by significant increases in relative frequency of single- or tandem C→T transitions at pyrimidine dinucleotides. Please click here to view a larger version of this figure.
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 genes1,2,25. The λ Select cII assay is based on the retrieval of the λLIZ shuttle vectors from the genomic DNA of cells/tissues of the transgenic animals, followed by packaging of the rescued vectors into λ phage heads that can infect an appropriate host E. coli. Subsequently, the infected bacteria are grown under selective conditions to allow for scoring and analysis of mutations in the cII transgene (see Figure 1)1,3. The λ Select cII assay has been used extensively for mutagenicity testing of a wide range of chemicals and/or physical agents (reviewed in references2,49). The assay has been successfully applied to transgenic mouse/rat cell cultures treated in vitro with various chemicals and/or physical agents, and the tissues/organs of the corresponding animals treated in vivo with different test chemicals/agents4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,34,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75.
The λ Select cII assay in cultured cells of transgenic rodents treated with a test compound represents, in many ways, a viable alternative to in vivo mutagenicity experiments in the corresponding animals treated with the tested chemical/agent3. As a general rule, the in vitro models offer significant advantages over their counterpart in vivo animal models, as they are much less labor intensive and costly, require far less time to be completed, and most importantly, do not involve the direct use of the animals2,50,52. At the same time, the in vitro models may not fully recapitulate all aspects of mutagenesis due to differences in the pharmacokinetic and pharmacodynamic properties of chemicals between the cultured cells in vitro and experimental animals in vivo2,3. For example, chemicals whose route of exposure is inhalation (e.g., cigarette smoke or electronic cigarette vapor) can only be made amenable to in vitro testing in cell cultures after they are converted from gaseous or vapor forms to liquid or condensate, which complicates their pharmacokinetics. Also, an incomplete or absent metabolic capacity of cultured cells in vitro to convert certain chemicals into DNA-reactive species may not represent DNA-damage driven mutagenicity in animals exposed in vivo to genotoxic chemicals2,3. Although, this drawback may be compensated for, to varying extents, by the addition of an external metabolic activation system (i.e., S9 mix) to the in vitro cell culture models22.
Furthermore, the replication of real life human exposure to genotoxic chemicals/agents is more limited with in vitro cell culture models than with experimental animals in vivo3. Generally, humans are exposed to chronic doses of genotoxic agents over a span of several years to a few decades76,77,78. The finite lifespan of cells in culture, as compared to the relatively longer lifetime of rodents (i.e., days/weeks versus a few years) makes modeling of human exposure to genotoxins more challenging in the former models2,3. Nonetheless, mutagenicity analysis with in vitro cell culture models can provide an initial indication of the genotoxic potential of a given chemical/agent(s), and the results can be used as a guide to design 'refined' in vivo experiments which feature a 'reduced' number of animals2,3.
In conclusion, the λ Select cII assay in cultured cells of transgenic rodents treated with a test compound, or the corresponding animals treated with the tested chemical/agent, is a valuable approach for mutagenicity testing. We have successfully used the approach, as have other research groups throughout the world4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,34,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75. More recently, we have expanded the applications of this approach by developing a new technique in which a modification of the λ Select cII assay together with next-generation sequencing enables high throughput analysis of mutations in a time-, cost-, and labor-effective manner23.
The authors have nothing to disclose.
We would like to acknowledge the contributions of all colleagues and collaborators to our original studies, whose results have been referred to in this manuscript (for illustrative purposes). The authors' work is supported by grants from the National Institute of Dental and Craniofacial Research of the National Institutes of Health (1R01DE026043) to AB and from the University of California Tobacco-Related Disease Research Program to AB (TRDRP-26IR-0015) and ST (TRDRP-25IP-0001). The sponsors of the study had no role in study design, data collection, data analysis, data interpretation, writing of the report, or in the decision to submit for publication.
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 |