Construction of eukaryotic expression vector of mouse microRNAs miR-22

Objective To clone microRNA22 fragment from human genomic DNA and construct it into the eukaryotic expression vector.Methods MicroRNA22 genomic sequence was amplified from mouse genomic DNA by PCR and cloned into pUCm-T plasmid,then sequenced.The sequence was cloned into eukaryotic expression vector pIRES2-EGFP plasmid,then identified with BamHⅠ and EcoRⅠ in 1.5% agarose gel electrophoresis and sequenced.Hela cells were tansfected with the identified pIRES2-EGFP/miR-22 and pIRES2-EGFP by Lipofectamin 2000.Results The 334-bp genomic fragment amplified by PCR was correct.Eukaryotic expression vector pIRES2-EGFP/miR-22 was successfully constructed and confirmed by sequencing.G418-resistant clones of Hela cells were obtained and confirmed as positive by RT-PCR.Northern blotting showed the expression of miR-22 increased.Conclusion The successful cloning of genomic sequence corresponding to pre-miR-22 and construction of its eukaryotic expression vector has laid the foundation for the further study of miR-22 function.