The Assay

The transgenic mice are given repeated doses of the suspected carcinogen for a week or two. If the chemical is mutagenic, it will cause random mutations throughout the genome of each mouse cell. If a mutation occurs in either

• the lacI gene (which encodes the lac repressor) or
• the operator,

the gene (lacZ) for beta-galactosidase will no longer be repressed. To detect this,

• The DNA is extracted from the tissues of the treated mouse.
• The vector is isolated and used to make functional bacteriophages.
• E. coli cells are mixed with the bacteriophage and spread on a solid culture medium.
• The bacteriophages infect and destroy ("lyze") the E. coli cells.
• This causes clear circular zones, called plaques, to appear in a "lawn" of bacteria.
• Before they die, cells that have been infected by bacteriophages carrying a mutated lacI or operator will produce beta-galactosidase.
• This reacts with a substrate in the culture medium turning it blue.
• Bacteriophages with unmutated genes produce colorless plaques because no beta-galactosidase is synthesized.
• Count both colorless and blue plaques.
• The number of blue plaques divided by the total number of plaques gives the mutation frequency.

The graph shows the results of a test done on the spleen cells of a transgenic mouse that had been dosed with benzopyrene, a known mutagen (and carcinogen). Three days after its last dose, 46 blue plaques were recovered from the spleen sample out of a total of 2,647,040 plaques. This yields a mutation frequency of 1.7 x 10⁻⁵ (1.7 in 100,000) — a significant increase over control values.

This photograph (courtesy of Stratagene) shows one mutant (blue) plaque on a lawn of E. coli containing many non-mutant (clear) plaques.