Development of efficient RNA interference system using EGF-displaying phagemid particles
Abstract
Aim: To develop an efficient RNA interference system using phagemid particles displaying the epidermal growth factor (EGF) ligand.
Methods: pSilencer1.0-siEGFP and pSilencer4.1-siAkt plasmids were constructed by gene clone technology. The modified helper phage genome (plasmid) M13KO7EGFCT was used to package phagemids, such as pSilencer1.0-siEGFP and pSilencer4.1-siAkt. ELISA was used to quantify the titer of the progeny virus particles. Single-strand DNA was extracted and analyzed by agarose gel electrophoresis to evaluate the percentage of the phagemid particles. The expression level of the reporter gene enhanced green fluorescence protein (EGFP) was determined by transducing phagemid particles packaging pSilencer1.0-siEGFP into cells. The level of Akt gene expression in cells transduced phagemid particles packaging pSilencer4.1-siAkt was examined by Western blotting. Hydroxycamptothecin (HCPT) was used to enhance the gene transduction efficiency.
Results: RNAi vectors pSilencer1.0-siEGFP and pSilencer4.1-siAkt were successfully constructed. Phagemid-encoding siRNA can be packaged efficiently. After the cells were infected by EGF displaying phagemid particles in the presence of HCPT, the expression of the target gene EGFP or Akt was substantially downregulated.
Conclusion: Cell-targeted phagemid particles are efficient siRNA delivery vectors in the presence of HCPT.
Keywords:
Methods: pSilencer1.0-siEGFP and pSilencer4.1-siAkt plasmids were constructed by gene clone technology. The modified helper phage genome (plasmid) M13KO7EGFCT was used to package phagemids, such as pSilencer1.0-siEGFP and pSilencer4.1-siAkt. ELISA was used to quantify the titer of the progeny virus particles. Single-strand DNA was extracted and analyzed by agarose gel electrophoresis to evaluate the percentage of the phagemid particles. The expression level of the reporter gene enhanced green fluorescence protein (EGFP) was determined by transducing phagemid particles packaging pSilencer1.0-siEGFP into cells. The level of Akt gene expression in cells transduced phagemid particles packaging pSilencer4.1-siAkt was examined by Western blotting. Hydroxycamptothecin (HCPT) was used to enhance the gene transduction efficiency.
Results: RNAi vectors pSilencer1.0-siEGFP and pSilencer4.1-siAkt were successfully constructed. Phagemid-encoding siRNA can be packaged efficiently. After the cells were infected by EGF displaying phagemid particles in the presence of HCPT, the expression of the target gene EGFP or Akt was substantially downregulated.
Conclusion: Cell-targeted phagemid particles are efficient siRNA delivery vectors in the presence of HCPT.