Cre/lox-mediated Site-specific Recombination In Zebrafish Embryos

Conventional transgenic approaches result in random genome integration oftransgenes, alimitationthat diminishtheirusefulness infishdirectional breeding.Theuse of gene-targeting methodologies is desirable to control the expression, efficiencyof insertion, and locus of incorporation of transgenes into fish genomes. Crerecombinase-mediated cassette exchange (RMCE) is an efficient and convenient newmethod of gene site-direct integration. Our experiments demonstrated that RMCEbased on Cre/lox system can function efficiently and accurately in the zebrafishsystem. And we established a primary gene targeting platform in transgenic fish. Thefollowingistheintroducitontoeachchaper.Chapter 1: Review the limitations of conventional transgenic approaches, theresearch on gene targeting in fish genetic engineering, and the use of Cre/lox systemintransgenicfield,especiallyintransgeniczebrafish.Chapter 2: Construction of two flox recombinant plasmids used in Crerecombinase mediated cassette exchange: pCMV_lox66CreintEGFP_lox2272 andp_lox71RFP_lox2272.Chaper 3: Zebrafish's zygotes were co-injected with capped Cre RNA and twoflox recombinant plasmids. Red fluorescence, genomic PCR analysis and DNAsequencing showed that RFP gene in p_lox71RFP_lox2272replaced the flox DNA segmentin pCMV_lox66CreintEGFP_lox2272. These results demonstrated that RMCE based onCre/loxsystemcanfunctioninthezebrafishsystem.Chaper 4: Atransgenic zebrafish line, harboring a target site and expressing crerecombinase, was generated by using conventional transgenic method. The filialgeneration , second of target zebrafish line ,was injected with p_lox71RFP_lox2272 to makeflox gene RFP replace the genomic flox gene. Three zebrafish with red fluorescencewere obtained. Genomic PCR analysis and DNA sequencing confirmed the correctgene target events. This site-directed integration system using RMCE based onCre/lox system should be a promising gene targeting platform serving various...