The Effect Of Recombinant Adenovirus-Mediated Sirna On Adiponectin Expression In The 3T3-L1 Cells

PART 1THE CONSTRUCTION OF RECOMBINANT ADENOVIRUS EXPRESSING MICE ADIPONECTIN ACRP30 SIRNA BY HOMOLOGOUS RECOMBINATION IN BACTERIAOBJECTIVE: To construct the RNA interference adenoviral vector specific for Adiponectin (Acrp30) gene.METHODS: The plasmids(pSilencer1.0-U6-Acrp30-1, 3 and pSilencer1.0-U6-GFP) and the shuttle vector (pShuttle) were digested with endonuclease separately. The shuttle plasmids ( pShuttle-U6-Acrp30-1, pShuttle-U6-Acrp30-3, and pShuttle-GFP) were linearized by Pme I and transformed into BJ5183-AD-1 cells (harbor pAdEasy-1) by electroporation. Positive clones were selected by Kanamycin-resistant colonies and confirmed by DNA miniprep and PacI digestion. Plasmids from correct clones were amplified by transforming into XL10-Gold cells. Again, miniprep plasmid DNA was made by a standard alkaline lysis procedure. The resulting adenoviral DNA (pAd-U6-Acrp30-1, pAd-U6-Acrp30-3 and pAd-U6-GFP) were verified by PacI digestion analysis and DNA sequencing then transformed into XL10-Gold for large scale amplification. After amplificated in ultra-competent XL10-Gold, the recombinants are transformed into AD293 cells to package and amplify recombined adenovirus.RESULTS: The recombinant adenoviral vector with mice Acrp30 siRNA was constructed successfully. The titers were 4.5×108;3.7×108;5.6×108 PFU/ml separately after amplification.CONCLUSION: The siRNA adenoviral vector against Acrp30 expression has been successfully conctructed, readying for further reaserch. PART 2ACRP30 SIRNA INHIBIT THE ADIPONECTIN EXPRESSION IN ADIPOSE CELL LINESOBJECTIVE: To observe the effect of Acrp30 siRNA recombinant adenovirus on mice Acrp30 expression in 3T3-L cells METHODS:3T3-L1 cell line was transfected with the Acrp30-siRNAadenovirus expression vector. The mRNA expression and protein levels of Acrp30 in those cells were evaluated by semi-quantitative RT-PCR and ELISA.RESULTS: The mRNA expression of the Acrp30 gene in 3T3-L1 cells with adenovirus plasmids was statistically significantly lower than in control 3T3-L1 cells (P<0.001) as assessed by semi-quantitative RT-PCR. ELISA analysis showed that the Acrp30 expression was also efficiently droped at protein levels by 3T3-L1 cells transduced with Acrp30-siRNA adenovirus.CONSTRUCTION : The siRNA adenoviral vector against Acrp30 expression inhibits the expression of Acrp30 in 3T3-L1 cells successfully and effectively.