A High Efficient Gene Targeting In Multiple Locus Induced By Zinc Finger Nucleases

The researchers have been more and more caring about the technique of gene target which is gradually coming to be mature.However,the traditional gene target is merely relied on homologous recombination,whose efficiency is just between 10^-6 and 10^-5.Recently,we have managed to establish a technique of multiple locus on gene targeting using the middle repeat of the interval sequence of rDNA as a target,and succeeded to produce the transgenic mandarin fishes and the recombination cow fertilized egg cell.Although in these researches the efficiency of gene target has been up to 200-300 folds,it is still a long way to real high efficiency. According to recent research,so many techniques about gene target with high efficiency have been put into use at home and abroad,in which a famous method is the application of zinc finger nuclease that can high improved the efficiency of gene target.Our research has combined the application of zinc finger nuclease with the multiple loci gene target,which can improved the efficiency of gene target up to 18%in theory.This article has used the ITS1 between 18S and 5.8S of rDNA as a target,and got a pair of three-zinc finger gene cutting the target sequence which combined with the coding sequence of Fok I slicing domain,thus we got a pair of zinc finger nucleases gene.And then,we inserted each zinc finger nuclease gene into the eukaryotic expression vector of pcDNA3.1-NLS,and got a pair of pcS-ZFNs.It was inserted into the pMD19-T vector for the construction of the recombinant vector pMD19-DS1-pCMV-EGFP-DS2 that the pCMV-EGFP gene which amplified by PCR using pEGFP-N1 out of MCS as template and two homogenous recombination arms of DS1 and DS2 which were amplified from human genome by PCR.By cotransfecting a pair of pcS-ZFNs and recombinant vector into HEK293 cells,we got the gene targeting cells with stable growth and reproduction,and persistent expression of the green fluorescence.After extracting the genome DNA of former HEK293 cells and testing the gene target by PCR,we managed to test the recombination efficiency of the gene target which is up to 20%.This research can avoid the bad reaction of the drug,improve the efficiency of gene target without drug selection and provide a transgenic technique for the transgenic animal and the gene therapy and diagnostics of human.