Abstract 353: Rapid Gene Editing in Rat Models of Human Disease Using Targeted Nucleases
Previous investigations into mechanisms of genes in animal models have primarily used the mouse because of the availability of technologies for targeted manipulation of its genome in embryonic stem (ES) cells. To accelerate functional mechanistic studies in other species where ES technology is not yet widespread, we recently developed an alternative and rapid method for generating targeted mutations in rat genes by the application of Zinc Finger Nucleases (ZFNs). When introduced into an embryo, ZFNs target and induce a chromosome double strand DNA break at the investigator-specified locus, stimulating cellular responses which can result in knock out or knock in modifications at the locus. Using commercially available reagents, we targeted and disrupted more than 100 protein-coding genes across 13 different inbred, consomic, and outbred genetic backgrounds with a 97% (102/105) success rate in approximately 30 months. ZFNs can therefore access nearly every gene in the rat genome and can induce multiple different alleles at the target locus. The majority of mutations, 81% (355/437) were simple deletions ranging from 1-228 base pairs (median 13-bp) and upon breeding, 94% (178/189) of mutations were transmitted to the next generation with little evidence of germline mosaicism. Many of these strains are now available for functional mechanistic studies of candidate human disease genes for hypertension and chronic kidney disease. Co-introduction of a homologous gene template with the ZFNs could stimulate knockin at three different loci in the rat genome. Gene knock into the rat Rosa26 locus allows for uniform expression of a gene in all cells and tissues of the adult animal as well as germline-competent ES cells derived from the Fawn Hooded Hypertensive strain. This knockin technology in embryos and ES cells offers many new opportunities to make very precise modifications to the rat genome and is a key step toward creating conditional gene alleles in the rat. Transcriptional activator-like effector nucleases (TALENs) are also active in the rat embryo and we have disrupted 70% (7/10) genes with this new technology. In sum, targeted nuclease technology is enabling many new genetic approaches to the rat and other model systems to create even better human disease models.
- © 2012 by American Heart Association, Inc.