PMID: 20650762Jul 24, 2010Paper

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

Nan fang yi ke da xue xue bao = Journal of Southern Medical University
Xue-hu XuWei-ming Li

Abstract

To clone hsa-miR-148a and construct its retroviral expression vector. 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. 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. 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.

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