Construction Of TALEN Vector For Jmjd3 Gene Site-specific Editing In Human And The Effects Of Jmjd3 Knockout In Human Cervical Cancer Cells In Vitro

Objectives:To construction of TALEN vector for jmjd3 gene editing in human by TALEN technology and observe the effects of jmjd3 knockout on the Growth in human cervical cancer cells in vitro. Methods:Construction of TALEN vector for jmjd3 gene editing in human(TALEN left arm plasmid L1 and TALEN right arm plasmid R1).The two plasmids were identified by endonuclease and then sequenced. TALEN left arm plasmid L1, TALEN right arm plasmid R1 and EGFP fluorescence plasmid transfected totally to human cervical cancer cell line Hela with normal Hela cells acting as blank control group. The efficiency of transfection is calculated after 24 hours and then screened using puro solution(2ug/ul) to all cell death in blank control group(>3d). The remaining cells in transfection group continued to cultivate.jmjd3 m RNA were detected by RT-PCR;MTT assay was performed to examine cell proliferation. Results:Verified by restriction enzyme cleave identification, and further confirmed by DNA sequencing and Blast comparative analysis. the TALEN plasmids for jmjd3 gene editing in human were constructed successfully. We transfected TALEN plasmid into Hela cells. In comparison with the control group, the level of jmjd3 m RNA was down-regulated dramatically by TALEN plasmids targeting jmjd3 showing a down-regulation of m RNA by(46.5±2.0)%; The activity of cell proliferation in experimental group was obviously inhibited, which was detected by MTT assay(P <0.05). Conclusion:1. The artificial TALEN plasmid targeting jmjd3 of human can edit the jmjd3 gene loci of human.2、The proliferation abilities of human cervical cancer cell line Hela were significantly suppressed after jmjd3 gene editing,which shows that jmjd3 participates in proliferation regulation of cervical cancer cell.