| Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin | |
| Wu, Yuping; Zhang, Guangxian; Xiong, Qian; Luo, Fang; Cui, Caimei; Hu, Wei; Yu, Yanhong; Su, Jin; Xu, Anlong; Zhu, Zuoyan; Xu, AL, Sun Yet Sen Zhongshan Univ, Coll Life Sci, State Key Lab Biocontrol, Open Lab Marine Funct Genom State High Tech Dev, Guangzhou 510275, Peoples R China | |
| 2006-06-01 | |
| Source Publication | MARINE BIOTECHNOLOGY
 |
| ISSN | 1436-2228 |
| Volume | 8Issue:3Pages:304-311 |
| Abstract | Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.; Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F(1) generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P(0) generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism. |
| Subtype | Article |
| Keyword | Gh Gene Integration Knockin Knockout Zebrafish |
| Department | Sun Yet Sen Zhongshan Univ, Coll Life Sci, State Key Lab Biocontrol, Open Lab Marine Funct Genom State High Tech Dev, Guangzhou 510275, Peoples R China; Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China |
| Subject Area | Biotechnology & Applied Microbiology ; Marine & Freshwater Biology |
| DOI | 10.1007/s10126-006-5116-7 |
| WOS Headings | Science & Technology ; Life Sciences & Biomedicine |
| Indexed By | SCI |
| Language | 英语 |
| WOS Research Area | Biotechnology & Applied Microbiology ; Marine & Freshwater Biology |
| WOS Subject | Biotechnology & Applied Microbiology ; Marine & Freshwater Biology |
| WOS ID | WOS:000238157700009 |
| WOS Keyword | BACTERIAL ARTIFICIAL CHROMOSOMES ; INSERTIONAL MUTAGENESIS ; STEM-CELLS ; ES CELLS ; GENES ; ANTISENSE ; CHIMERAS ; CLONING |
| Citation statistics | |
| Document Type | 期刊论文 |
| Identifier | http://ir.ihb.ac.cn/handle/152342/8954 |
| Collection | 期刊论文 |
| Corresponding Author | Xu, AL, Sun Yet Sen Zhongshan Univ, Coll Life Sci, State Key Lab Biocontrol, Open Lab Marine Funct Genom State High Tech Dev, Guangzhou 510275, Peoples R China |
| Affiliation | 1.Sun Yet Sen Zhongshan Univ, Coll Life Sci, State Key Lab Biocontrol, Open Lab Marine Funct Genom State High Tech Dev, Guangzhou 510275, Peoples R China 2.Chinese Acad Sci, Inst Hydrobiol, State Key Lab Freshwater Ecol & Biotechnol, Wuhan 430072, Peoples R China |
| Recommended Citation GB/T 7714 | Wu, Yuping,Zhang, Guangxian,Xiong, Qian,et al. Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin[J]. MARINE BIOTECHNOLOGY,2006,8(3):304-311. |
| APA | Wu, Yuping.,Zhang, Guangxian.,Xiong, Qian.,Luo, Fang.,Cui, Caimei.,...&Xu, AL, Sun Yet Sen Zhongshan Univ, Coll Life Sci, State Key Lab Biocontrol, Open Lab Marine Funct Genom State High Tech Dev, Guangzhou 510275, Peoples R China.(2006).Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin.MARINE BIOTECHNOLOGY,8(3),304-311. |
| MLA | Wu, Yuping,et al."Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin".MARINE BIOTECHNOLOGY 8.3(2006):304-311. |
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