To further increase TK mediated tumor killing efficacy and

facilitate tracing TK expression, we constructed a new vector by inserting a CMV enhancer and an EGFP reporter gene into pGL3-hTERTp-TK vector, and evaluated its therapeutic efficacy in in vitro and in vivo tumor therapy. Materials and methods 1. Reagents Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing click here company. PCR kit and TaqMan real time PCR kit were from Takara Bio-engineering Co., Ltd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma, USA; Ganciclovir (GCV) was from ROCH company. Lipofectamine 2000, DMR IE2C and Trizol were from Invitrogen. TRAPEZE® RT telomerase activity detection kit Akt targets was purchased from KeyGen (Nanjing, China). Plasmid Midi Kit

was from Heda Biotech (Guangzhou, China). All PCR primers were synthesized by Shanghai Ying-Jun Biotechnology Co., Ltd. 2. Cell lines Human nasopharyngeal carcinoma 5-8F cells (NPC 5-8F), human breast cancer cells MCF-7 GW2580 mouse and human vascular endothelial cells ECV were kindly provided by Department of Cell Biology, the Southern Medical University, and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 incubator (Shell LAB, USA) as previously reported [10]. 3. Construction of plasmid with luciferase reporter gene EGFP gene was obtained from pEGFP-N1 by PCR using forward primer Egfp-F: CCCAAGCTTATGGTGAGCAAGGGCGAGGAG and reverse primer Egfp-R: GCTCTAGATTACTTGTACAGCTCGTCCATGC. 406 bp CMV enhancer fragment was obtained from pEGFP-N1 by PCR using forward primer hCMVen-F: 5-CGGGATCCCGCGTTACATAACTTACGGT-3′ and reverse primer hCMVen-R: 5-ACGCGTCGACCAAAACAAACTCCCATTGAC-3. Miconazole 1131 bp TK gene with NCBI accession number AY575228 was obtained from pMD18-TK by PCR using forward primer 5-CCGCTCGAGATGGCTTCGTACCCCTGC-3′ and reverse primer 5-CCCAAGCTTGTTAGCCTCCCCCATCTC-3. The 260 bp hTERT promoter was obtained from pMD18-T-hTERTp using forward primer hTERTp-F: 5-GGGGTACCAGTGGATTCGCGGGCACAGACG-3′ and reverse

primer hTERTp-R: 5-CCGCTCGAGAGGGCTTCCCACGTGCGCAGCA-3. All PCR fragments were verified by DNA sequence analysis. Stop codon TGA of TK gene was removed in TK reverse primer to facilitate the construction of TK-EGFP fusion protein. EGFP fragment was digested with Hind III and Xba I and subcloned into pGL3-basic plasmid to obtain pGL3-basic-EGFP. TK fragment was excised with Hind III and Xho I and subcloned into pGL3-basic-EGFP to construct pGL3-basic- TK-EGFP. hTERTp fragment was subcloned into pGL3-basic-TK-EGFP at Kpn I and Xho I sites to construct pGL3-basic-TK-hTERTp-EGFP. CMV enhancer fragment was inserted into pGL3-basic-TK-hTERTp-EGFP at BamH I and Sal I site according to previous reports [11, 12] to construct the enhanced vector pGL3-basic-hTERTp-TK- EGFP-CMV. All plasmids were verified by restriction enzyme digestion. 4.