Tokgun Onur, Fiorentino Francesco Paolo, Tokgun Pervin Elvan, Yokota Jun, Akca Hakan
Department of Medical Biology, Pamukkale University School of Medicine, Denizli, Turkey.
Kitos Biotech srls, Porto Conte Ricerche, 07041, Alghero, Italy.
Mol Biotechnol. 2017 Jun;59(6):200-206. doi: 10.1007/s12033-017-0006-y.
Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345. Induction of MYC expression by doxycycline was detectable in both cell lines by real-time PCR and western blot analysis. This study highlights the relevance of pTIJ vector to allow the inducible expression of any gene of interest. In our belief, pTIJ will be an extremely useful tool to simplify the generation of genetically engineered cell lines for the inducible expression of cDNA sequences in biological studies. Furthermore, we report the generation of a pTIJ-MYC vector for the inducible expression of the oncogene MYC.
慢病毒载体是基因表达研究的有力工具。在此，我们报告了pTIJ的构建，这是一种用于诱导基因表达的载体。pTIJ由pTRIPZ骨架构建而来，pTRIPZ原本设计用于诱导shRNA序列的表达，通过在Tet启动子上游引入多克隆位点并去除miR30侧翼序列而得到。为了评估pTIJ作为诱导表达目的基因工具的效果，我们将MYC cDNA导入pTIJ，并感染了两种小细胞肺癌细胞系H209和H345。通过实时PCR和蛋白质印迹分析，在两种细胞系中均检测到强力霉素诱导的MYC表达。本研究突出了pTIJ载体对于诱导任何目的基因表达的相关性。我们认为，pTIJ将是一种极其有用的工具，可简化在生物学研究中用于诱导cDNA序列表达的基因工程细胞系的构建。此外，我们报告了用于诱导癌基因MYC表达的pTIJ-MYC载体的构建。
