Cholesterol is synthesized in chicken through de novo lipid biosynthetic pathway where two most important genes viz. SREBP1 and ACACA play immense role. To minimize cholesterol synthesis, RNAi approach was adopted and accordingly, we developed transgenic chicken possessing ACACA and SREBP1 shRNA constructs, which showed lower level of ACACA and SREBP1 in serum. The serum total cholesterol, triglycerides, HDL and LDL cholesterol was significantly lower by 23.8, 35.6, 26.6 and 20.9%, respectively in SREBP1 transgenic birds compared to the control. The egg total cholesterol and LDL cholesterol content was numerically lower in both ACACA and SREBP1 transgenic birds by 14.3 and 13.2%, and 10.4 and 13.7%, respectively compared to the control. It is concluded that the protocol was perfected to develop transgenic chicken through RNAi for knocking down the expression of ACACA and SREBP1 proteins, which minimized the cholesterol and triglycerides contents in serum and eggs.
Stearoyl-CoA desaturase (SCD) is key enzyme in the production of triglycerides and cholesterol. Present study was conducted to know the expression pattern of the Stearoyl-CoA desaturase (SCD) gene in liver during juvenile age (0 day, 14th day, 28th day and 42nd day) in layer birds. Expression of SCD gene increased from the day of hatch to 42nd day, as the body weight and fat deposition increased. The mRNA expression levels of SCD gene during day 0 was differed significantly (P less than 0.05) with expression levels during other juvenile ages. When compared with SCD expression levels on the day 0 expression levels were up-regulated by 8198.64, 7245.99 and 5587.77 folds over day 14, 28 and 42 respectively. Serum concentration of triglycerides and cholesterol were found to be proportional to the expression levels of SCD gene in liver. It is concluded that SCD expression varied among different age groups during juvenile stages and had significant association with serum triglycerides and cholesterol concentration in layer chicken.
RNA interference by short hairpin RNAs (shRNAs) 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 involved in denovo lipid biosynthesis were designed upon considering parameters such as secondary structures of shRNAs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target), synthesized and cloned in pENTR/U6 entry vector to knockdown the expression of SCD and SREBP1 genes. After transfection of these shRNA constructs into the chicken embryonic hepatocytes, expressions of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes was observed in hepatocytes. The shRNAs against SCD gene showed the knock down efficiency ranged from 20.4% (shRNA5) to 74.2% (shRNA2). In case of SREBP1 gene, the shRNAs showed knock-down efficiency ranging from 26.8% (shRNA4) to 95.85% (shRNA1). The shRNAs against both the genes introduced in chicken hepatocyte cells did not show any significant impact on expression of immune response genes (IFNA and IFNB) in those cells. These results clearly demonstrated the successful down regulation of the expression of SCD and SREBP1 genes by the shRNA molecules against both the target genes under in vitro condition. It is concluded that the shRNA molecules against SCD and SREBP1 genes showed great potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cells.
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 (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 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.
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