Prediction andin vitrosilencing of two lipid biosynthetic genes viz.SCDandSREBP1in chicken using RNA interference

Abstract

AbstractRNA interference is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each againstSCDandSREBP1genes were designed after considering the parameters like secondary structures of shRNA constructs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target) to knockdown their expression. After successful transfection of these shRNA molecules into the chicken embryonic hepatocyte culture, expression of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression ofSCDandSREBP1genes with minimal activation of immune response genes in hepatocytes was observed after transfecting the shRNA molecules into them. The shRNA constructs againstSCDgene showed the knock down efficiency ranged from 20.4% to 74.2%. In case of shRNA constructs againstSREBP1gene, they showed knock down efficiency ranging from 26.8% to 95.85%. These results clearly demonstrated the successful down regulation of the gene expression by designed shRNA molecules against both the target genes underin vitrocondition. The shRNA2 molecule forSCDgene and the shRNA1 molecule forSREBP1gene were found as the best among all the shRNA molecules used for silencing the target genes under cell culture system. It is concluded that the shRNA molecules designed against SCD and SREBP1 genes showed potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cell culture system.