[Cloning of hsa-miR-148a and construction of its retroviral expression vector].

ABSTRACT

OBJECTIVE: To clone hsa-miR-148a and construct its retroviral expression vector. METHODS: The pre-miR-148a amplified by PCR was inserted to pMSCV to construct the recombinant retroviral expression plasmid pMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148a and PIK packaging plasmid were cotransfected into 293FT packaging cells by calcium phosphate-mediated transfection to produce the retrovirus, and the retrovirus titer was measured by infection of NIH3T3 cells. RESULTS: Restriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed successfully, and the virus titer was 5x10(8) CFU/ml after infection of NIH3T3 cells. CONCLUSION: The successful construction of the retroviral expression vector MSCV-miR-148a allows the production of high-titer retrovirus to facilitate further study of the molecular functions of miR-148a.