Design and Construction of Bidirectional Tumor-Specific Expression Vector to Silence or Over-expressSimultaneously in Cancer Gene Therapy

Due to the drawbacks of small interfering RNA, and/or non-specific and low promoter activityof vector-based RNA polymerase III promoters, need to find and develop a novel therapy incancer. To specify and enhance the expression of vector-based RNAi, the tumor-specificpromoters are concerned. Survivin, a member of apoptosis inhibitor family, is exhibitingexclusive potential properties in cancer gene therapy. The aim of present study is to design anddevelop the survivin promoter in bidirectional, which is able to silence or over-express twogenes simultaneously. Promoter of survivin as RNA polymerase II promoter was elected. Usingbioinformatics tools, the core promoter sequence of survivin was selected and designed in twodirections. The 1100 nucleotide fragment was synthesized in GenScript, and subsequentlycloned in pRNAT-U6.1/Neo expression vector (6380 bp) as background. The substitution ofthe U6 promoter into the bidirection survivin core promoter was evaluated with doubledigestion analysis. To assess the efficiency of promoter activity, two shRNA oligonucleotideswas inserted in the downstream of each promoter and the QRT-PCR assay was conducted toestimate the mRNA expression level. The construction of tumor-specific expression vectorwith two shRNA (6888 bp) was verified by double digestion test. The expression of mRNAwas significantly reduced approximately 54% after treatment. The current recombinant vectoras an expression vector-based RNA polymerase II promoter can be applied for silencing and/orover-expression of two target genes. Hope to overcome the cancer via novel gene therapytechniques.

کلیدواژه ها:

Survivin ، bidirection promoter ، RNA polymerase II & III ، cancer gene therapy