generation of knockout rats with x-linked severe combined immunodeficiency (x-scid) using zinc-finger nucleases一代的基因敲除大鼠与x连锁使用锌指核酸酶重度联合免疫缺陷症(x).pdfVIP

Generation of Knockout Rats with X-Linked Severe Combined Immunodeficiency (X-SCID) Using Zinc-Finger Nucleases 1 1 1 1 2 1 Tomoji Mashimo *, Akiko Takizawa , Birger Voigt , Kazuto Yoshimi , Hiroshi Hiai , Takashi Kuramoto , Tadao Serikawa1 1 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2 Shiga Medical Center for Adult Disease, Moriyama, Japan Abstract Background: Although 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 Findings: We 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 Significance: The 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 to