Methodology Of Injection From Portal Vein The Hepcidin Gene-specific RNAi Adenovirus In Mouse

Objective:To construct a RNAi combinant adenoviral expressive vectors pAD-U6-shRNA-CMV-EGFP containing mouse hepcidin gene,and and to observe their gene silencing effect.Materials and Methods:(1) The construction of pAD-U6-shRNA-CMV-EGFP:According to the sequence of hepcidin gene in genbank,design the appropriate siRNA,Architecture oligo,synthesized shRNA sequence.Designing and synthesizing a pair of complementary oligomeric single-stranded DNA containing shRNA sequences.using special annealing systerm to form a double-stranded DNA,then the recombinant clones was obtained by BLOCK-iT U6of RNAi the Entry Vector Kit (Invitrogen,Catalog nos.K4944-00).insert the double-stranded shRNA oligo into shRNA expression vectors pENTR/U6,constructed shRNA expression plasmid according to a certain connection systera.Connection product was transformed into competent cells and multiplied. Plasmid was extracted and sequenced. Use designed Primers and Specific digestion system to Transform pENTR/U6Vector by Connecting to the CMV promoter and fluorescent protein (GFP),recombinant with pAd/BLOCK-IT-DEST by LR recombination system of Invitrogen, recombinant product was transformed into competent cells and multiplied.Plasmid was Filter and sequenced. after digested with Pac I,recombinant product was transfected into293A cells,fhen virus was Packaged,collected the primary virus liquid, the secondary amplification, and the collection of virus stock titer was determined standby.(2) the recombinant virus was infused into portal vein in mouse:after normal balb/c mouse was anesthesiaed,2*10e12vp/500ul virus was injected into portal vein,the mouse was Normal feeded in10d,then the liver specimens was taken down,hepcidin gene expression in mouse liver was detected by semiquantitative PT-PCR method.Results:After gene sequencing,the expression vector was constructed sucessfully, and the result of the plasmid sequencing contained the Target gene. a titer of2.7109ifu/ml adenosis venom was obtained.after the portal vein injection,the recombinant pAD-U6-shRNA-CMV-EGFP could down-regulate the mRNA levels of hepcidin expression in mouse liver,Clearly showied the inhibitory effect.Conclusion:a RNAi combinant adenoviral expressive vectors pAD-U6-shRNA-CMV-EGFP containing mouse hepcidin gene is constructed successfully.hepcidin expression in mouse liver could be down-regulated efficiently by the RNAi adenovims infused through portal vein.