Abstract

RNA-interference is the mechanism of sequence-specific, post-transcriptional gene silencing, initiated by small interfering RNA (siRNA), homologous to the gene being suppressed. Several techniques are utilized to transfer siRNA into cultured cells or animal models, while every method has advantages and disadvantages. In this study, a siRNA expression recombinant adeno-associated virus (AAV) was established by inserting H1 promoter into transfer plasmid of AAV Helper-Free system. To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and added into HEK293 cells, respectively. The results proved the expression of EGFP and p53 in cells were definitely suppressed at 72 h post-infection, which suggested that the H1 promoter, inserted into the recombinant AAV, could express siRNA in mammalian cells and this siRNA delivery system could be used for long-term gene silencing.

Key words: Adeno-associated virus, RNA interference, p53, EGFP.

Abbreviation

RNAi, RNA interference; siRNA, small interfering RNA; AAV, adeno-associated virus; ITRs, inverted terminal repeats; RISC, RNA-induced silencing complex; FACS, fluorescence activated cell sorting; MCS, multiple cloning site; qPCR, quantitative PCR; DAB, diaminobenzidine; shRNA, short hairpin RNA; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CMV, cytomegalovirus; qPCR, quantitative polymerase chain reaction.