Construction 3′-UTR of NOTCH1 dual luciferase recombintant vector and evaluation of its activity

Objective:To construct the dual-luciferase recombinant vector which contain 3′-UTR of NOTCH1 gene and verify its activity.Methods:3′-UTR of NOTCH1 gene was amplified through PCR method,and was inserted in the dual luciferase reporter vector which was digested by enzyme.3′-UTR of NOTCH1 which targeting effect miRNA was predicted by bioinformation.HEK293T cells was treated with recombinant vector or empty vector and miR-34a inhibitor or control by transfection reagent.The dual Luciferase Reporter Assay System was used to evaluate the activity of luciferase.Results:3′-UTR of NOTCH1 gene was successfully cloned into the pmiR-RB-REPORTTM vector,which was vertified by Gel electrophoresis and DNA sequencing methods.3′-UTR of NOTCH1 gene may has a binding site of miR-34a.Comparing with HEK293T cells was treated with the empty vector,the luciferase activity of HEK293T cells which was cotransfected with recombinant vector and miRNA-34a inhibitor was increased to 45%.Conclusion:3′-UTR of NOTCH1 gene dual luciferase combintant vector was successfully constructed.preliminary evidence show that miR-34a could regulate expression of NOTCH1.