-
Epstein-Barr virus (EBV) is a human gamma herpes virus closely associated with nasopharyngeal carcinoma (NPC). Among the twelve proteins encoded by the EBV genome, the latent membrane protein-1(LMP1) gene has been a focus of interest as a target gene due to its ability of transform certain rodent fibroblast cell lines (3). Therefore, triggering RNAi (RNA interference), by the introduction of siRNA (small interfering RNA) aimed at the LMP1 gene, into NPC cells to knockdown LMP1 gene expression holds promise as a gene therapy approach for control of NPC.
siRNA is most commonly achieved in stable form by using shRNA (short hairpin RNA) expressed from a DNA vector or virus (5, 6, 8, 10) with a polymerase-Ⅲ U6/H1 RNA promoter. We designed the LMP1 gene-specific insert (pshLMP1 expression cassette) such that it specifies a 19nt sequence derived from the target LMP1 transcript, separated by a loop of four oligonucleotides from the reverse complement of the same 19nt sequence (1). The pshLMP1 expression cassette was incorporated according to the traditional "annealing method" (1) and then cloned into the pTZU6+1 vector between the U6 promoter and T5 termination and checked by sequencing. In our previous research, deletion and mutant of nucleotides in pshLMP1 expression cassette were frequently observed when the "annealing method" was implemented, which may severely restrict its application in RNAi technology. We therefore established an alternative and novel "PCR method" to construct pshLMP1 vectors and demonstrated it has greater efficiency compared to the annealing method, and can facilitate the effective application of shRNA vectors for antiviral purposes.
The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV
- Received Date: 05 July 2006
- Accepted Date: 24 December 2006
Abstract: To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.