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Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites
Liu, Wei-Yi; Wang, Yun; Qin, Yao; Wang, Ya-Ping; Zhu, Zuo-Yan; Zhu, ZY, Wuhan Univ, Chinese Acad Sci, Grad Sch, Inst Hydrobiol,State Key Lab Freshwater Ecol & Bi, Wuhan 430072, Peoples R China
2007
Source PublicationMARINE BIOTECHNOLOGY
Volume9Issue:4Pages:420-428
AbstractWith current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.
KeywordCre-lox Gene Integration Site-specific Recombination Transgenic Zebrafish
DepartmentWuhan Univ, Chinese Acad Sci, Grad Sch, Inst Hydrobiol,State Key Lab Freshwater Ecol & Bi, Wuhan 430072, Peoples R China
Subject AreaBiotechnology & Applied Microbiology ; Marine & Freshwater Biology
Indexed BySCI
Language英语
Document Type期刊论文
Identifierhttp://ir.ihb.ac.cn/handle/152342/8412
Collection期刊论文
Corresponding AuthorZhu, ZY, Wuhan Univ, Chinese Acad Sci, Grad Sch, Inst Hydrobiol,State Key Lab Freshwater Ecol & Bi, Wuhan 430072, Peoples R China
Recommended Citation
GB/T 7714
Liu, Wei-Yi,Wang, Yun,Qin, Yao,et al. Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites[J]. MARINE BIOTECHNOLOGY,2007,9(4):420-428.
APA Liu, Wei-Yi,Wang, Yun,Qin, Yao,Wang, Ya-Ping,Zhu, Zuo-Yan,&Zhu, ZY, Wuhan Univ, Chinese Acad Sci, Grad Sch, Inst Hydrobiol,State Key Lab Freshwater Ecol & Bi, Wuhan 430072, Peoples R China.(2007).Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites.MARINE BIOTECHNOLOGY,9(4),420-428.
MLA Liu, Wei-Yi,et al."Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites".MARINE BIOTECHNOLOGY 9.4(2007):420-428.
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