Prediction and in vitro silencing of two lipid biosynthetic genes viz. SCD and SREBP1 in chicken using RNA interference

Abstract

RNA interference is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes were designed after considering the parameters like secondary structures of shRNA constructs, mRNA target regions, GC content and thermodynamic properties ({Delta}G overall, {Delta}G duplex and {Delta}G 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 of SCD and SREBP1 genes with minimal activation of immune response genes in hepatocytes was observed after transfecting the shRNA molecules into them. The shRNA constructs against SCD gene showed the knock down efficiency ranged from 20.4% to 74.2%. In case of shRNA constructs against SREBP1 gene, 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 under in vitro condition. The shRNA2 molecule for SCD gene and the shRNA1 molecule for SREBP1 gene 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.