In Vivo Mammalian Mutagenesis

Environmental Health Sciences

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Abstract

Somatic and germinal mutations can have a severe impact on the fitness of multicellular organisms and offspring. The ability to study the occurrence of mutations in higher organisms has been limited by available methods rendering it difficult to address important questions such a 1) kinetics of mutation induction in relation to organ specificity, 2) mutation induction during development, 3) systemic effect of somatic mutations of age-related degenerative diseases, and 4) tumor progression from pre-neoplastic to neoplastic growth in relation to genomic stability. Transgenic systems based on recoverable vectors are the only systems that can be used to study the induction of mutagenicity in all organs. Unfortunately the spontaneous mutant frequency is 10 to 20 fold higher than in endogenous genes. Mutations in transgenic systems can originate from three different sources of DNA damage. Type 1: Damage in the DNA that is caused and fixed in the animal (in vivo). Type 2: Damage in the DNA that has occurred either in the animal or artificially during preparation of the DNA and later fixed in bacterial cells during recovery of the phage (in vitro). Type 3: Spontaneous mutations arising during the first replications of non-mutant phages under selective conditions (in vitro). A PhiX174 transgenic cell line PX-2, derived from a homozygous embryo of a PhiX174 transgenic mouse was used to develop the approach for separation of the three sources of mutations. The origin of the three classes of mutations can be distinguished by using the single burst assay (SBA). Type 1 revertants (in vivo) have a burst size (BS) >55, type 2 (DNA damage, in vitro fixation) have 13