title: A promoter trap vector for knocking out bovine myostatin gene with high targeting efficiency.
abstract: 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.
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: L H
authors: LH
authors: Zhao
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: Y H
authors: YH
authors: Zhao
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: H
authors: H
authors: Liang
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: T
authors: T
authors: Yun
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: X J
authors: XJ
authors: Han
authors: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China.
authors: M L
authors: ML
authors: Zhang
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: X
authors: X
authors: Zhou
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: D X
authors: DX
authors: Hou
authors: State Key Laboratories of Reproductive Medicine, Nanjing Medical University, Nanjing, China lirongfeng@njmu.edu.cn.
authors: R F
authors: RF
authors: Li
authors: The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology, Inner Mongolia University, Hohhot, China.
authors: X L
authors: XL
authors: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005333:Fetus; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009132:Muscles; D055435:Myostatin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D055435:Myostatin; D049452:Green Fluorescent Proteins; D019868:Kanamycin Kinase; D004720:Endonucleases