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

Author: QinYao
Tutor: ZhuZuoYan;WangYaPing
School: Institute of Hydrobiology
Course: Genetics
Keywords: Cre / lox system Recombinase-mediated cassette exchange Site-specific integration Zebrafish
CLC: Q78
Type: Master's thesis
Year: 2006
Downloads: 178
Quote: 1
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Abstract


The random integration of the exogenous gene prevents the traditional transgenic approaches in the the the fish orientation breeding field of application . The use of gene targeting technology is expected to achieve site-specific integration of exogenous genes : control of exogenous gene integration sites , the integration rate and the temporal and spatial expression . Cre / lox site-specific recombination mediated cassette exchange reaction is a simple and efficient Gene targeting new method . This study demonstrated the feasibility of this method in fish , and zebrafish as a model, initially established a site-specific integration technology based on the Cre / lox system of fish . This article consists of five chapters : Chapter I : Summary of the limitations of the traditional transgenic approaches , the development of the fish gene targeting technology , Cre / lox system in transgenic applications and applications in the zebrafish research status . Chapter II : Cassette exchange reaction carrier . Constructed a two flox recombinant plasmid : the target vector pCMV lox66 < / sub > of CreintEGFP lox2272 and targeting vector p the lox71 < / sub > RFP lox2272 < / sub > . Chapter 3 : coinjection of Cre RNA and two flox recombinant plasmid into zebrafish fertilized egg , the emergence of the red fluorescence and PCR detection and sequencing results showed that the RFP reporter gene targeting vector efficient replacement lox sites in the target carrier DNA fragments . Confirmed the Cre / lox system mediated cassette exchange reaction in zebrafish feasibility . Chapter 4: traditional transgenic approaches to create a turn pCMV lox66 < / sub > of CreintEGFP lox2272 < / sub > of zebrafish strains , the strains containing high-level expression of exogenous gene target and self - expression of Cre enzymes. Targeting the vector p lox71 the RFP lox2272 < / sub > < / sub > injected into the F2 fertilized egg successfully screened three red fluorescent zebra fish . PCR and sequencing further confirmed the cassette exchange reactions . As a result, the initial establishment of fish site-specific integration technology based on the Cre / lox system .

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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