This project aims to rapidly produce rat mutants through a transposon-based method of mutagenesis. The rat is a favored model for many types of human disease for which mice are not suitable. The rat is the most relevant model for cardiovascular and hypertension disease, with a heart rate much more similar to that of humans compared to mice. As opposed to the mouse, rats and humans also share a more similar number of cytochrome P450 genes, perhaps explaining why the rat has been a more useful model for toxicology and pharmacology studies. The rat is also a favored model for diabetes, arthritis, behavioral disorders (including drug addiction), and brain imaging. Unlike the acrocentric mouse genome, the rat and human genomes are predominantly metacentric, making chromosomal comparisons more relevant for modeling human genetic diseases. However, rat mutants cannot be easily generated through traditional methods of homologous recombination in embryonic stem (ES) cells, because rat ES cells cannot be cultured or manipulated. Alternative methods are inefficient and costly. While the rat has not had the ease of genetic manipulation that the mouse has had over the past twenty years, it certainly possesses physiological, anatomical, and chromosomal similarities to humans that make for a more relevant model of human disease. Now, this proposal seeks to rapidly produce rat mutants through the development of hyperactive transposons for germ line insertional mutagenesis. This approach uses randomly integrating transposons, which enables the rapid identification of sequence-tagged mutation sites.
The Aims will focus on synthesizing hyperactive transposases from three different families of transposons, each with unique insertion characteristics. The current standard for mutagenesis in rodents using DNA transposons is a rate of 1-3 insertions per gamete in rats and mice. However, to be commercially viable and produce at least one null allele in each offspring, this transposition rate must be substantially improved. Our goal is to increase the mutation frequency to a level at least 25X greater than the transposons currently used for rodent mutagenesis. Hyperactive transposases will be the direct product from this study, while the ultimate Phase II goal will be the generation of rat mutants (MutaRats) for modeling human disease. Offspring of MutaRats (MutaRat Knockout Rat Models) will not be phenotyped or mated to produce a breeding colony, but sperm will be isolated from mutant males and cryogenically frozen for future retrieval and fertilization using already existing technology. Mutant animals will be distributed by the National Rat Resource and Research Center and shared with the academic community according to NIH policies for sharing model organisms for biomedical research.
About 89% of all compounds tested for drug use fail during the final stages of approval due to unacceptable side effects or a lack of efficacy. There is a clear need to screen drugs more effectively in animals during preclinical testing before entering the most expensive phases of drug testing in humans. This project is designed to employ a new method using mobile DNA elements (or """"""""jumping genes"""""""") for the rapid and economical production of a large variety of mouse and rat models of human disease, which will enable a greater scrutiny of candidate drugs and will facilitate more favorable testing in humans.
