Jan A. Gossen

Spontaneous and X-ray-induced deletion mutations in a LacZ plasmid-based transgenic mouse model

Transgenic mouse mutation models carrying bacterial marker genes in bacteriophage lambda shuttle vectors have been applied to study spontaneous or induced mutations in vivo. However, due to the nature of the shuttle vector these models are insensitive to large deletions. Clastogenic agents, which predominantly induce large deletions, were therefore found to yield very low responses in these assays. Here we report the use of LacZ plasmid-based transgenic mice, allowing the detection of a broad spectrum of mutations. Treatment of mice with X-rays (5 x 50 rads) resulted in induction of up to about 5-fold higher mutation frequencies in lung, spleen and liver. Analysis of spontaneous and induced mutant LacZ genes indicated that at least 40-50% of all mutations were caused by deletions. The possibility of detecting a broad spectrum of mutations with this system suggests that the LacZ plasmid-based transgenic mouse may be the mammalian model of choice for studying spontaneous and induced mutations in vivo.

Transgenic mice as model systems for studying gene mutations in vivo

Transgenic mice carrying bacterial reporter genes have been developed to study spontaneous or induced mutations in vivo. Mutations can be analysed upon the efficient retrieval of reporter genes from mouse genomic DNA into a suitable bacterial host. These systems allow, for the first time, the direct correlation of mutational mechanisms in vivo with their ultimate physiological endpoints, for example, cancer or ageing.

LacZ transgenic mouse models : their application in genetic toxicology

Gene mutations have been implicated in the etiology of cancer, developmental anomalies, genetic disease and aging. Many different methods for mutation detection have been developed and applied to obtain a more fundamental insight in the chain of molecular events that ultimately lead to mutations. Most of these methods, however, can only be applied to cultured cells and therefore do not allow comparative analysis of mutations in various organs and tissues in an intact organism. The main difficulty in studying mutagenesis in chromosomal DNA is to identify and isolate mutated genes with a high efficiency. Here we describe the development and application of LacZ transgenic mouse models for studying, in different organs and tissues, spontaneous or induced mutations. Such models allow study of the induction of DNA damage, repair, mutagenesis and carcinogenesis in one animal system. Accordingly, results obtained may ultimately provide greater insight into the chain of events from in vivo exposure to genotoxic agents to mutations and their ultimate physiological endpoints. In addition to their use in fundamental research, transgenic animal mutation models find a major application in the field of genetic toxicology testing, in particular with respect to organ specificity.

Use of transgenic mouse models for studying somatic mutations in aging

Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested. Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging. The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used. Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed. One conclusion is that, due to the nature of the transgene, lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations. To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene. Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging.

New Methods for Detecting DNA Sequence Variation in Relation to Aging

The availability of accurate and sensitive methods for the detection of DNA sequence variation is an important prerequisite for obtaining insight in the genetics of aging. DNA sequence changes in somatic cells are considered as causal factors in aging, while the accumulation in the germ line of late-acting deleterious mutations could underly the genetic component of age-related disorders, such as Alzheimer disease. We have taken two new experimental approaches for studying DNA sequence variation in genomic DNA from higher animals. First, we have used transgenic mice harboring a number of tandemly integrated shuttle vectors with a lacZ bacterial marker gene for measuring both spontaneous and induced mutations in various organs and tissues. Second, by using a two-dimensional electrophoretic separation system (size-separation in neutral gels, followed by denaturing gradient polyacrylamide gel separation on the basis of sequence content) we have been able to detect minute variations in minisatellite and simple sequences. The use of these novel experimental systems in studying the molecular basis of aging will be discussed.