Abstract:Objective: To construct a new dual fluorescent protein reporter gene plasmid, psiCHECK-2-eGFP-mLumin. Methods: The dual fluorescent protein reporters gene plasmid was constructed by inserting the PCR amplification products of enhanced green fluorescent protein (eGFP) and mLumin into psiCHECK-2 vector to replace the firefly luciferase and renilla luciferase, respectively. The sequence of eGFP and mLumin were confirmed by PCR, and blasted online of the National Center for Biotechnology Information. HEK293T cells were transfected with psiCHECK-2-eGFP-mLumin plasmid and the expression efficiency of the fluorescent proteins was valuated. Results: The dual fluorescent protein reporters gene plasmid, psiCHECK-2-eGFP-mLumin, was successfully constructed by inserting the correct sequence of eGFP and mLumin into the psiCHECK-2 plasmid. The psiCHECK-2-eGFP-mLumin successfully mediated the gene expression of eGFP and mLumin in HEK293T cells. Conclusion: The psiCHECK-2-enhanced green fluorescent protein-mLumin plasmid was constructed successfully and expressed well in HEK293T cells.
[1]Amodio N, Di Martino MT, Neri A, et al. Non-coding RNA: a novel opportunity for the personalized treatment of multiple myeloma[J]. Expert Opin Biol Ther, 2013, 13(Suppl 1): S125-137.[2]Lee HW, Jeon YH, Hwang MH, et al. Dual reporter gene imaging for tracking macrophage migration using the human sodium iodide symporter and an enhanced firefly luciferase in a murine inflammation model[J]. Mol Imaging Biol,2013. [Epub ahead of print].[3]陈伟, 陈翀, 张焕新, 等.小鼠adam10基因启动子克隆和双荧光素酶报告基因系统构建及鉴定[J]. 中国实验血液学杂志, 2012, 20(3): 740-743.[4]Luna C, Li G, Qiu J, et al. Role of miR-29b on the regulation of the extracellular matrix in human trabecular meshwork cells under chronic oxidative stress[J]. Mol Vis, 2009, 15: 2488-2497.[5]Prsumey J, Salzano G, Courties G, et al. PLGA microspheres encapsulating siRNA anti-TNF alpha: efficient RNAi-mediated treatment of arthritic joints[J]. Eur J Pharm Biopharm, 2012, 82(3): 457-464.[6]Tarver JE, Sperling EA, Nailor A, et al. miRNAs: small genes with big potential in metazoan phylogenetics[J]. Mol Biol Evol, 2013. [Epub ahead of print].[7]Liu SG, Qin XG, Zhao BS, et al. Differential expression of miRNAs in esophageal cancer tissue[J]. Oncol Lett, 2013, 5(5): 1639-1642.[8]Kubowicz P, Zelaszczyk D, Pekala E. RNAi in clinical studies[J]. Curr Med Chem, 2013, 20(14): 18011816.[9]Chu J, Zhang Z, Zheng Y, et al. A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions[J]. Biosens Bioelectron, 2009, 25(1): 234-239.[10]Jung G, Zumbusch A. Improving autofluorescent proteins: comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants[J]. Microsc Res Tech, 2006, 69(3): 175-185.[11]Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP)[J]. Gene, 1996, 173(1 Spec No): 33-38.[12]Novello C, Pazzaglia L, Cingolani C, et al. miRNA expression profile in human osteosarcoma: role of miR1 and miR133b in proliferation and cell cycle control[J]. Int J Oncol, 2013, 42(2): 667-675.[13]Abbasi M, Lavasanifar A, Uludag H. Recent attempts at RNAi-mediated Pglycoprotein downregulation for reversal of multidrug resistance in cancer[J]. Med Res Rev, 2013, 33(1): 33-53.[14]Lee JY, Kim S, Hwang do W, et al. Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation[J]. J Nucl Med, 2008, 49(2): 285-294.[15]Yun B, Anderegg A, Menichella D, et al. MicroRNA-deficient Schwann cells display congenital hypomyelination[J]. J Neurosci, 2010, 30(22): 7722-7728.[16]毕延震,郑新民,邵长伟,等.检测miRNA 活性的双荧光素酶单载体[J]. 中国生物化学与分子生物学报, 2012, 28(8):775-780.