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Abstract ΔFosB, a truncated form of FosB, which impacts on fat and calcium deposition, drug addiction and other aspects, has extensive biological functions. The objective of this study was to silence the expression of ΔFosB using the RNA interference by recombinant adenovirus, and provided a material to investigate the relative functions of ΔFosB. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-492/572) which targeting two different sites of ΔFosB mRNA and then oligonucleotides were cloned into shuttle vector pENTR/CMV-GFP/U6. After detection of interference efficiency, we recombined a pENTR/CMV-GFP/U6-572 and adenovirus backbone vector pAD/PL-DEST, to produce recombinant vector pAD/PL-DEST/CMV- GFP/U6-572. The fifth generation recombinant adenovirus particles (AD-ΔFosB-572) were produced and further amplified by transfecting HEK-293 cells. The titer of adenovirus reached 1.58 × 109 PFU/mL determined by TCID50 assays. Western blot and Real-time Quantitative PCR indicated that AD-ΔFosB-572 had better interference efficiency by infecting goat mammary gland epithelial primary cells after 24, 48 and 72 h, in which mRNA expression levels of ΔFosB gene were reduced 45%, 73% and 81%， respectively. Western blot also showed that the expression of ΔFosB protein was reduced by infecting the cells with AD-ΔFosB-572. So AD-ΔFosB-572 has been proved that it has significant interference effect on expression of ΔFosB and can be used for subsequent studies of gene function.
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Received: 21 April 2010
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