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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We investigated the pathogenesis of chronic allograft rejection in mouse cardiac allografts. Long-term survival occurred after administration of monoclonal antibody to CD4 or CD40-ligand (CD40L) plus donor cells. Both treatments induced permanent graft survival, but, in contrast to transplants in mice treated with CD4 monoclonal antibody, grafts in mice treated with CD40L monoclonal antibody lacked evidence of chronic rejection, including transplant arteriosclerosis. Freedom from chronic rejection in the group treated with CD40L monoclonal antibody correlated with vascular expression of the 'protective' genes heme oxygenase-1 (HO-1), Bcl-xL and A20. Moreover, arteriosclerosis was induced in allografts in immunoglobulin-deficient mice by antibody transfer only when the transfer was done before expression of protective genes. A direct role for protective gene expression in endothelial cells was demonstrated by in vitro experiments in which induction of HO-1 or Bcl-xL suppressed alloantibody-stimulated endothelial activation. Finally, induction of HO-1 in vivo protected allografts against chronic injury. These data show a role for protective genes in the prevention of chronic rejection, and indicate new approaches to protect grafts against development of transplant arteriosclerosis.

A direct role of carbon monoxide (CO), an effector-signaling molecule during heme oxygenase-1 (HO-1) catalysis of heme, in the protection against hepatic ischemia/reperfusion (I/R) injury needs to be established. This study was designed to determine the effects and downstream mechanisms of CO on cold I/R injury in a clinically relevant isolated perfusion rat liver model. After 24 hours of cold storage, rat livers perfused ex vivo for 2 hours with blood supplemented with CO (300 parts per million) showed significantly decreased portal venous resistance and increased bile production, as compared with control livers perfused with blood devoid of CO. These beneficial effects correlated with improved liver function (serum glutamic oxaloacetic transaminase levels) and diminished histological features of hepatocyte injury (Banff's scores). The CO-mediated cytoprotective effects were nitric oxide synthase-and cyclic guanine monophosphate-independent, but p38 mitogen-activated protein kinase (MAPK)-dependent. Moreover, adjunctive use of zinc protoporphyrin, a competitive HO-1 inhibitor, has shown that exogenous CO could fully substitute for endogenous HO-1 in preventing hepatic I/R insult. This study performed in a clinically relevant ex vivo cold ischemia model is the first to provide the evidence that HO-1-mediated cytoprotection against hepatic I/R injury depends on the generation of, and can be substituted by, exogenous CO. The p38 MAPK signaling pathway represents the key downstream mechanism by which CO prevents the I/R insult. In conclusion, regimens that employ exogenous CO should be revisited, as they may have potential applications in preventing/mitigating I/R injury, and thus expanding the liver donor pool for clinical transplantation. (HEPATOLOGY 2002;35:815-823.)

The genetic dissection of physiological and pathological traits in laboratory model organisms is accelerated by the ability to engineer loss-of-function mutations at investigator-specified loci. This chapter describes the use of zinc-finger nucleases (ZFNs) for the targeted disruption of endogenous rat genes directly in the embryo. ZFNs can specifically disrupt target genes in cultured rat cells and in embryos from inbred and outbred strains, leading to permanently genetically modified animals. This technology allows for the rapid, targeted modification of the rat genome.