Zhao L H, Zhao Y H, Liang H, Yun T, Han X J, Zhang M L, Zhou X, Hou D X, Li R F, Li X L
The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
Genet Mol Res. 2015 Mar 31;14(1):2750-61. doi: 10.4238/2015.March.31.5.
With the development of gene targeting approaches, genomic mutation technologies in livestock animals such as gene trapping, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats and their associated systems have been improved. Although ZFNs have been used for gene targeting in many species, the off-target sites are still present. Using gene trapping, the workload of screening of targeted clones was decreased by generating a smaller number of drug-resistant clones. Determining whether the efficiency of gene trapping is lower than that of ZFNs for a specific gene has been challenging. In this study, to knock out the bovine myostatin gene, we constructed a promoter trap vector and compared its efficiency with that of ZFNs. The promoter trap vector contained a green fluorescent protein sequence without the promoter and a neomycin phosphotransferase (neo(R)) cassette driven by the phosphoglycerate kinase promoter. When the trapping vector was inserted downstream of the endogenous promoter, the fluorescent protein gene was expressed. The targeted-positive cell clones were identified based on green fluorescence and G418 double selection, followed by polymerase chain reaction analysis and sequencing. The targeting efficiency reached 5%. Compared with the efficiency of ZFN pairs (5.17 and 2.86%), the promoter trap vector PIII-myostatin could knock out the bovine myostatin gene. Therefore, gene trapping may be an effective tool for genomic modification.
随着基因靶向方法的发展，家畜基因组突变技术如基因捕获、锌指核酸酶（ZFNs）、转录激活样效应核酸酶以及成簇规律间隔短回文重复序列及其相关系统已得到改进。尽管ZFNs已被用于许多物种的基因靶向，但脱靶位点仍然存在。使用基因捕获，通过产生数量较少的耐药克隆减少了靶向克隆的筛选工作量。确定基因捕获对于特定基因的效率是否低于ZFNs一直具有挑战性。在本研究中，为了敲除牛肌肉生长抑制素基因，我们构建了一个启动子捕获载体，并将其效率与ZFNs的效率进行比较。该启动子捕获载体包含一个无启动子的绿色荧光蛋白序列和一个由磷酸甘油酸激酶启动子驱动的新霉素磷酸转移酶（neo(R)）盒。当捕获载体插入内源性启动子下游时，荧光蛋白基因得以表达。基于绿色荧光和G418双重筛选，随后进行聚合酶链反应分析和测序来鉴定靶向阳性细胞克隆。靶向效率达到了5%。与ZFN对的效率（5.17%和2.86%）相比，启动子捕获载体PIII-肌肉生长抑制素能够敲除牛肌肉生长抑制素基因。因此，基因捕获可能是一种用于基因组修饰的有效工具。
