Construction and Identification of lentiviral Expression Vector of MiR-218

To construct a lentiviral vector expressing Hsa-pre-mir-218-2 and make a foundation on studying of the functions and mechanisms of miR-218.partially complementary forward and reverse primers were designed based on the hsa-pre-mir-218-2 sequence.The primer dimers were generated by primer annealing method.These primer dimers were amplified by PCR and were inserted into enzyme-digested and linearized pGCSIL-GFP lentiviral vectors.The recombinant plasmid pGCSIL-GFP-mir-218-2 was identified by double enzyme digestion and then was packed with lentiviral packaging systems,and viral titer was determined.Human gastric cancer cells MKN-28 were infected with the constructed lentiviral vectors.The expression of miR-218 in MKN-28 cells was determined by qPCR.The double enzyme digestion and DNA sequencing analyses of the recombinant plasmid revealed that insertion element was correctly cloned into the vector.The qPCR demonstrated that the expression level of miR-218 in the lentivirus infected MKN-28 cells was increased significantly.It can effectively infect gastric cancer cells.The lentiviral vector with hsa-pre-mir-218-2 is constructed successfully and laid an experimental basis on the studies of the functions and mechanisms of miR-218.