Screening for porcine Sn gene-specific amiRNAs

In order to construct a specific artificial miRNA (amiRNA) expression vector targeting the porcine sialoadhesin (Sn) gene and to screen for effective amiRNAs, the 1147-2280 bp sequence of the porcine Sn cDNA was amplified by RT-PCR and inserted into the eukaryotic expression vector pEGFP-N1 to construct the reporter vector pSn-EGFP for amiRNA screening. Ten amiRNAs targeting the porcine Sn gene were designed and the corresponding recombinant interfering plasmids (pcDNA5-miRSn-110) were constructed. NIH-3T3 cells were co-transfected with the reporter vector and the interfering vectors and the expression of the sequence-specific amiRNAs was assessed by poly(A)-tailed RTPCR. Fluorescent microscopy and FACS analysis revealed that all ten Sn gene-specific amiRNAs expressed from the vectors inhibit Sn-EGFP fusion protein levels. Of these ten amiRNAs, the highest inhibition efficacies were observed for amiRSn3, amiRSn8 and amiRSn9 which inhibited Sn protein expression by 95.37%, 91.27% and 84.7% respectively. These findings suggest that effective amiRNAs identified by this screening can be used to construct adenovirus amiRNA expression vectors targeting the Sn gene.