Journal Article

Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases

Zhang, Zhiying, Zhang, Cunfang, Xu, Kun, Ren, Gang, Zhang, Tingting, Wang, Ling, Li, Zhanwei, Ren, Chonghua

2014

Overview

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 Fok I 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.

• Publisher: Springer Netherlands,Springer Nature B.V
• Subject: Animal Anatomy / Animal Biochemistry / Animals / Animals, Genetically Modified / Base Sequence / Biomedical and Life Sciences / cold / Endonucleases - biosynthesis / Endonucleases - genetics / fibroblasts / flow cytometry / gene targeting / Genes / Genes, Reporter / Genetic engineering / Green Fluorescent Proteins - biosynthesis / Green Fluorescent Proteins - genetics / HEK293 Cells / Histology / Humans / Life Sciences / mice / Morphology / mutants / Myostatin - genetics / nucleases / probability / Protein Engineering / rats / Sequence Analysis, DNA / Sheep / Sheep, Domestic - genetics / Zinc / Zinc Fingers