College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, Xianyang, 712100, Shaanxi, People's Republic of China.
Mol Biol Rep. 2014 Jan;41(1):209-15. doi: 10.1007/s11033-013-2853-3. Epub 2013 Nov 6.
Prior to the development of zinc-finger nuclease technology, genetic manipulation by gene targeting achieved limited success in mammals, with the exception of mice and rat. Although ZFNs demonstrated highly effective gene targeted disruption in various model organisms, the activity of ZFNs in large domestic animals may be very low, and the probability of identifying ZFN-mediated positive targeted disruption events is small. In this paper, we used the context-dependent assembly method to synthesize two pairs of ZFNs targeted to the sheep MSTN gene. We verified the activity of these ZFNs using an mRFP-MBS-eGFP dual-fluorescence reporter system in HEK293T cells and, according to the expression level of eGFP, we obtained a pair of ZFNs that can recognize and cut the targeted MSTN site in the reporter vector. The activity of ZFN was increased by cold stimulation at 30 °C and by mutant the wildtype FokI in ZFN with its counterpart Sharkeys. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two MSTN mutant cell lines, identified by flow cytometry and sequencing, were obtained.
在锌指核酸酶技术发展之前，除了小鼠和大鼠之外，通过基因打靶进行遗传操作在哺乳动物中取得的成功非常有限。虽然 ZFN 在各种模式生物中表现出高效的基因靶向敲除，但 ZFN 在大型家畜中的活性可能非常低，并且鉴定 ZFN 介导的阳性靶向敲除事件的概率很小。在本文中，我们使用基于上下文的组装方法合成了两对针对绵羊 MSTN 基因的 ZFN。我们使用 mRFP-MBS-eGFP 双荧光报告系统在 HEK293T 细胞中验证了这些 ZFN 的活性，并且根据 eGFP 的表达水平，我们获得了一对能够识别和切割报告载体中靶向 MSTN 位点的 ZFN。30°C 的冷刺激和突变野生型 FokI 及其互补 Sharkeys 可以提高 ZFN 的活性。最后，将 ZF-Sharkeys 和报告载体共转染到绵羊胎儿成纤维细胞中，通过流式细胞术和测序鉴定获得了两个 MSTN 突变细胞系。
