Identification Of Pseudo AttP Sites For The Streptomyces Phage φC31Integrase In Goat Genome

With the rapid development of transgenic technology,The transgenic animal safetymore and more people’s attention.ApplicationφC31integration mediated exogenous gene tothe efficient integration of directional integration,continued high expression to producetransgenic animals.Recent years already have been found in human, mouse, rat, Drosophila,cattle, zebrafish and so on,φC31integration mediated site-specific integration of exogenousgene into unmodified genome.At present,φC31integration mediated exogenous gene efficientintegration on goats as well as the existence of pesudo attP sequence has not been reported ingoat genome. This experiment detected the pseudo attP site in goat genome,Draw theintegrated map,Pick out the species-specific, high-frequency integration, expression of highlevels of integration sites used in the preparation of transgenic goats. The results are asfollows:1. we cloned Construction of a expression System for The Streptomyces phage φC31integrated: In this study, the main constructing the Streptomyces phageφC31integrase fusionexpression vector pCMV-myc-φC31.Goat fetal fibroblasts transfected byelectroporation.Collected and extracted protein,by Western Blotting. Detected the eukaryoticexpression ofφC31integrase in the goat fetus fibroblasts.The results show ed that Integrasewith the myc tag fusion protein was70.4KD,It was showed that φC31integrase could beexpressed in goat fetal fibroblasts.2. Construction of the reporting system and integrase system:In this study, usedmultiplex PCR amplification, restriction enzyme digestion, connection and transforming toconstructing the reporting vector pEGFP-C1-attB and the integrase expression vectorpCMV-φC31-int,pCMV-φC31-NLS-int,pCMV-φC31-3NLS-int.3. Screening the cell:In this study, through the reporter vector pEGFP-C1-attB andpCMV-φC31-int,pCMV-φC31-NLS-int,pCMV-φC31-3NLS-int transfected by electroporationin Goat fetal fibroblasts. After G418selection for transgenic cell lines stablytransfected.Extraction goat fetal fibroblasts monoclonal genome,used PCR to detecting attBsequence is broken, in order to exclude random integration.Detection attB sequence results showed that the part of the monoclonal happened random integration, a little monoclonalhappened site-specific integration.4. Identification and analysis of the pesudo attP site:φC31integrase-mediatedsite-specific integration is a directional integration.It was the form of a non-randomintegration integrated into the attP or the pseudo attP site,But specific integrated into the sitewas uncertain.The experiment used the most popular contemporary chromosome walkingtechniques to studying the known sequence adjacent unknown sequence,Detected the pseudoattP site in goat genome.The experiment according to the known sequence of pEGFP-C1-attB.It were designed three different higher annealing temperature specific primers,SP1,SP2,SP3.Provided with the kit four unique design of the annealing temperature lowerdegenerate primers to conducting thermal asymmetric PCR.To compare and analyze theresulting fragment with goat genome database.Experimental results show a pseudo attPintegration site CPSF1in the goat on chromosome4,Its occurrence frequency of3%(3/100).Another pseudo attP integration sites CpsM1is located on chromosome5in thegoat,Its occurrence frequency of2%(2/100).Cattle were found that the φC31integrasemediated site-specific integration to the pseudo attP site efficiency was much higher than inthe goat genome.φC31integrase mediated exogenous gene into the goat genome lessefficient,May be due to the intracellular protein interactions,Thereby inhibiting the φC31integrase activity, such as protein DAXX.