Construction of Lentiviral Vector for the Expression of Hsa-mir-196b

Objective To construct a lentiviral vector for the expression of Hsa-mir-196b. Methods Hsamir-196b gene amplified from the peripheral blood DNA of healthy subjects was inserted into the Lentilox 3.7 (pLL-3.7) plasmid by double digestion with HpaⅠ / XhoⅠand subsequent ligation. Plasmids pLL-3.7-mir-196b,pCMV-VSV-G and pCMV-dR8.91 were co-transfected into HEK-293FT cells for packaging of the lentivirus. HEK293FT cells were then transduced with an appropriately diluted lentivirus supernatant for the titration of virus titer. Expression of Hsa-mir-196b was investigated using real-time PCR. Results Results from the PCR and DNA sequencing confirmed that the recombinant plasmid pLL-3.7-mir-196b was successfully constructed. The titer of supernatant was 7.2 ±1.1× 107 TU / ml. The expression level of Has-mir-196b in the lentivirus transduced HEK-293FT cells was increased by 24-fold. Conclusion Hsa-mir-196b expression lentiviral vector was successfully constructed.