Abstract
BackgroundAlthough the rat is extensively used as a laboratory model, the inability to utilize germ line-competent rat embryonic stem (ES) cells has been a major drawback for studies that aim to elucidate gene functions. Recently, zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc.Methodology/Principal FindingsWe report here on ZFN-induced gene targeting of the rat interleukin 2 receptor gamma (Il2rg) locus, where orthologous human and mouse mutations cause X-linked severe combined immune deficiency (X-SCID). Co-injection of mRNAs encoding custom-designed ZFNs into the pronucleus of fertilized oocytes yielded genetically modified offspring at rates greater than 20%, which possessed a wide variety of deletion/insertion mutations. ZFN-modified founders faithfully transmitted their genetic changes to the next generation along with the severe combined immune deficiency phenotype.Conclusions and SignificanceThe efficient and rapid generation of gene knockout rats shows that using ZFN technology is a new strategy for creating gene-targeted rat models of human diseases. In addition, the X-SCID rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.
Highlights
Several strategies are available for producing a wide variety of genomic alterations in the mouse, the same cannot be said of the rat
The efficient and rapid generation of gene knockout rats shows that using zinc-finger nucleases (ZFNs) technology is a new strategy for creating gene-targeted rat models of human diseases
The X-linked severe combined immune deficiency (X-SCID) rats that were established in this study will be valuable in vivo tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies
Summary
Several strategies are available for producing a wide variety of genomic alterations in the mouse, the same cannot be said of the rat. Rat spermatogonial stem cells (SSC) have been isolated and cultivated in vitro but their yield proved unsatisfactory in terms of their ability to undergo homologous recombination [6,7] Besides these methods which are based on the in vitro genetic engineering of pluripotent stem cells, transposon-mediated mutagenesis [8] and Nethyl-N-nitrosourea (ENU) mutagenesis [9,10] have been used with some success for producing mutations in the rat genome. We recently reported on a high-throughput gene-driven strategy which uses the mutagen ENU and the Mu-transposition reaction (MuT-POWER) to rapidly detect induced mutations This was in addition to our investigation of intracytoplasmic sperm injection (ICSI) for recovering heterozygous genotypes of interest out of a large sperm cell repository [11,12]. Zinc-finger nucleases (ZFNs) were successfully used to create genome-specific double-stranded breaks and thereby induce targeted gene mutations in a wide variety of organisms including plants, drosophila, zebrafish, etc
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