The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. Using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or mRNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25-100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within four months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.The laboratory rat is a well-established model for the genetic dissection of human diseaserelated traits (1) despite the fact that targeted modification of its genome is largely intractable. We investigated the application of engineered zinc-finger nucleases (ZFNs;(2)) for the elimination of specific rat gene function and generation of "knockout" rats. ZFNs induce sitespecific, double-strand DNA breaks that can be repaired by the error-prone non-homologous end joining DNA repair pathway to result in a targeted mutation (Fig. 1A). In the fruit fly and zebrafish, direct embryo injection of ZFN-encoding mRNA has been used to generate heritable knockout mutations at specific loci (2).The design and validation of ZFN reagents to target a single-copy Green Fluorescent Protein (GFP) transgene inserted in a rat chromosome and two endogenous rat genes, IgM and

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Abstract:Mounting evidence has demonstrated that the autonomic system plays a role in the morbidity and mortality of certain cardiovascular disease states. Ventricular arrhythmias have been associated with the level of sympathetic activation. We attempted to determine if the presence of fibrosis, a marker for previous ischemic events, correlates with an increase in the number of left stellate ganglion nerve cell bodies which is indicative of hypersympathetic stimulation to the myocardial tissue. Left stellate ganglia were removed, sectioned and prepared using hematoxylin and eosin and Masson's trichrome stain. The interventricular septum of the heart corresponding to the stellate ganglion samples were removed, serially sectioned, and stained with hematoxylin and eosin and M asson's trichrome stain. The samples were described using a grading scale to quantify the percentage of fibrosis. Ganglion nerve cell bodies were then individually counted in three separate high-powered fields. A student's T-test was used to statistically evaluate the data. Stellate ganglions were sampled from 32 cadavers. Fibrosis was present within 72% (23/32) of the interventricular septums that were sampled. Nine interventricular septums were found to be free of fibrosis. For those interventricular septums that were positive for the presence of fibrosis, the mean left stellate ganglion nerve cell bodies was 39.8 (Range: 26-51). For those interventricular septums that were negative for the presence of fibrosis, the mean left stellate ganglion nerve cell bodies was . The difference between the mean nerve cell bodies for interventricular septums with fibrosis and without fibrosis was found to be statistically significant (P = 0.048). Histological changes in terms of the number of left stellate ganglion nerve cell bodies seem to be dependent upon the presence of fi brosis within the interventricular septum. Considering fibrosis of the interventricular septum is a marker for previous ischemic events, an increase in the number of nerve cell bodies of the left stellate ganglion in the presence of fibrosis suggests an association does exist between hypersympathetic stimulation to the myocardial tissue and myocardial infarction. Further research into this association is warranted in order to determine if left stellate ganglion blockade is a viable treatment option for arrhythmias following myocardial infarctions.

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