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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 PublicationMARINE BIOTECHNOLOGY
ISSN1436-2228
Volume8Issue:3Pages:304-311
AbstractProduction 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.
SubtypeArticle
KeywordGh Gene Integration Knockin Knockout Zebrafish
DepartmentSun 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 AreaBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
DOI10.1007/s10126-006-5116-7
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
Indexed BySCI
Language英语
WOS Research AreaBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
WOS SubjectBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
WOS IDWOS:000238157700009
WOS KeywordBACTERIAL ARTIFICIAL CHROMOSOMES ; INSERTIONAL MUTAGENESIS ; STEM-CELLS ; ES CELLS ; GENES ; ANTISENSE ; CHIMERAS ; CLONING
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Cited Times:10[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/8954
Collection期刊论文
Corresponding AuthorXu, 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
Affiliation1.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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