Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
PLoS One. 2010 Jan 25;5(1):e8870. doi: 10.1371/journal.pone.0008870.
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.
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 tools for evaluating drug treatment or gene therapy as well as model systems for examining the treatment of xenotransplanted malignancies.
尽管大鼠被广泛用作实验室模型，但由于无法利用具有种系能力的大鼠胚胎干细胞（ES 细胞），因此对于旨在阐明基因功能的研究来说，这是一个主要的缺陷。最近，锌指核酸酶（ZFNs）已成功用于创建基因组特异性双链断裂，从而在包括植物、果蝇、斑马鱼等在内的多种生物中诱导靶向基因突变。
方法/主要发现：我们在此报告了 ZFN 诱导的大鼠白细胞介素 2 受体γ（Il2rg）基因座的基因靶向，同源的人类和小鼠突变导致 X 连锁严重联合免疫缺陷（X-SCID）。将编码定制设计的 ZFN 的 mRNA 共注射到受精卵的原核中，可使具有多种缺失/插入突变的遗传修饰后代的产生率超过 20%。ZFN 修饰的创始者将其遗传变化与严重联合免疫缺陷表型一起忠实传递给下一代。
高效快速地产生基因敲除大鼠表明，使用 ZFN 技术是创建人类疾病基因靶向大鼠模型的新策略。此外，本研究中建立的 X-SCID 大鼠将是评估药物治疗或基因治疗的有价值的体内工具，也是检查异种移植恶性肿瘤治疗的模型系统。
