title: Targeted disruption of the sheep MSTN gene by engineered zinc-finger nucleases.
abstract: 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.
authors.affiliation: College of Animal Science and Technology, Northwest A&F University, No. 22 Xinong Road, Yangling, Xianyang, 712100, Shaanxi, People's Republic of China.
authors.forename: Cunfang
authors.initials: C
authors.lastname: Zhang
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Gang
authors.initials: G
authors.lastname: Ren
authors.forename: Zhanwei
authors.initials: Z
authors.lastname: Li
authors.forename: Chonghua
authors.initials: C
authors.lastname: Ren
authors.forename: Tingting
authors.initials: T
authors.lastname: Zhang
authors.forename: Kun
authors.initials: K
authors.lastname: Xu
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004720:Endonucleases / Q000096:biosynthesis / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D055435:Myostatin / Q000235:genetics*; D015202:Protein Engineering; D017422:Sequence Analysis, DNA; D034561:Sheep, Domestic / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: D055435:Myostatin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004720:Endonucleases