Preparation And Identification Of Recombinant Adenoviruses Carrying Short Hairpin RNA Targeting △FosB Of Goat

ΔFosB, a truncated form of FosB, impacts on fat and calcium deposition, drug addiction and other aspects. Many studies suggested thatΔFosB have a significant research value in human medicine which can promote bone formation and increase bone density. The objective of this study was to silence the expression ofΔFosB using the RNA interference by recombinant adenovirus, and to investigate the relative functions and mechanisms ofΔFosB in lactating mammary gland, the main results were as follows:(1) The Block-iTTM shRNA interference system of invitrogen was used in this experiment. According to the goatΔFosB sequence, we designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-492,shRNA-572) which targeting two different sites ofΔFosB mRNA, then oligonucleotides were cloned into shuttle vector pENTR/CMV-GFP/U6. The western blotting test ofΔFosB proteins in HEK-293 cells, cotransfected with pAdTrack-CMV-ΔFosB, has shown both pENTR/CMV-GFP/U6-492 and pENTR/CMV-GFP/U6-572 have interference effects, but effect of latter was better. The recombinant vector pAD/PL-DEST/CMV-GFP/U6-572, which can express the Short hairpin RNA ofΔFosB gene, produced by the LR reaction between recombinant vector pENTR/CMV-GFP/U6-572 and adenovirus backbone vector pAD/PL-DEST. The digestion of Pac I restriction enzyme and sequencing results shown the recombinant viral vector pAD/PL-DEST/CMV-GFP/U6-572 has constructed successfully.(2) Linear products of pAD/PL-DEST/CMV-GFP/U6-572, digested by Pac I restriction enzyme, was used for transfecting HEK-293 cells by Lipofectamine 2000. After 13 days, the first generation adenovirus were collected by repeated freezing and thawing when about 50% of cells began to appear cytopathic effect, then the fifth generation of AD-ΔFosB-572 adenovirus were obtained by repeated the processing 4 times. The titer of adenovirus reached 1.58×10~9 PFU/mL determined by TCID50 assays. We proved the optimum MOI of AD-ΔFosB-572 was 200 by infecting goat mammary gland epithetlial primary cells. Western blotting results indicated that AD-ΔFosB-572 has better interference efficiency in goat mammary gland epithetlial primary cells. The Real-time Quantitative PCR results corroborated its results and shown that AD-ΔFosB-572 infect goat mammary epithelial cells (MOI=200) after 24 h, 48 h and 72 h, the mRNA ofΔFosB gene expression levels were reduced 45%, 73% and 81%. AD-ΔFosB-572 has obvious interference effect and can be used in the further study of gene function.