Construction Of Vector Of Multiple Loci Gene Targeting In Siniperca Chuatsi

Transgenic techniques with random integration have extremely decreased the efficiency of integration, hereditation and expression of external genes in transgenic animals. However, the techniques of multiple loci gene targeting could be an effective approach to cover the above problem. In this process, the vector of multiple loci gene targeting was constructed . The vector would be crucial for the further research of the gene targeting techniques in vivo.First of all, Primers (HRDS11/HRDS12 and HRDS21/HRDS22) were designed to amplify homologous recombination directing sequences (HRDSl and HRDS2) using SP1416-T transporting plasmid as template. The gained fragments were cloned into pMD18-T plasmid. Secondly, HRDSl and HRDS2 one by one were cloned into general gene targeting vector of pCTKNeo to construct specific gene targeting vector of pCTKNeo-HRDS1/2 for Siniperca chuatsi . Afterwards, Primers (TF1/IF2) were designed to amplify hEFN gene fragment using pCDNA3-hIFNl plasmid as template, and then the gained fragment was cloned into specific gene targeting vector of pCTKNeo-HRDSl/2. Finally, The multiple loci gene targeting vector with hIFN gene for Siniperca chuatsi was constructed which was identified via PCR amplification, enzyme digestion and sequencing.