Construction Of LfcinB Gene Lentiviral Vector Driven By Dual-promoter And Its Effect On SKOV3 In Vitro

1 : Preliminary analysis on the promoter region of human MUC16 GeneAbstract: Objective To analyze the transcription start point and important regulatory regions of human MUC16 gene and determine core promoter of human MUC16 gene. Methods The promoter region of MUC16 gene was analyzed by bioinformatics. The cultured cell lines SKOV3 was cultured and its genomic DNA was extracted. The 594bp (-1844-1250/), 548bp (-1798/-1250), 326bp (-1576/-1250) and 170bp (-1420 / -1250) promoter fragments were produced and amplified by PCR, then PGEM-T-promoter recombinant plasmid were constructed and transformed into JM109 bacteria being competent cell, respectively. Then these promoter fragments were cloned into luciferase reporter gene (pGL3 ) vector, so PGL3-170, PGL3-326, PGL3-548 and PGL3-594 recombinant expression plasmid were constructed and transformed into JM109 bacteria being competent cell. With liposome-mediated transient transfection way, PGL3-basic-promoter expression plasmids were transient transfected into SKOV3 cells, and luciferase activities were measured in cells. Results The four expression systems carrying human MUC16 gene promoter fragment and luciferase reporter gene were successfully constructed, they were PGL3-170 (-1420/-1250), PGL3-326 (-1576/-1250), PGL3-548 (-1844/-1750 ) and PGL3-594 (-1798/-1750), respectively. These promoter fragments all had activities in SKOV3 cells. The 5 end of human MUC16 core promoter was 5 end of PGL3-326, and its 3 end was determined in the position of -1750 sequences. When 5 end moved from-1844 to -1420, promoter activity gradually increased, however the changes were little in magnitude. This suggests that there are not essential cis-acting elements in this sequence. Conclusion The four expression voctors carrying human MUC16 gene promoter fragment and luciferase reporter gene were successfully constructed. The core promoter of human MUC16 gene is determined.2: Two-promoter-driven lentiviral vector gene LfcinB ConstructionObjective To construct LfcinB lentivirus containing both MUC16 and HS_OVCOV1 promoters.Methods The targeted MUC16 promoter, HS_OVCOV1 promoter and LfcinB gene were connected to the lentiviral vector. The lentivirus MUC16 HS_OVCOV1-LfcinB unified plasmid with conditional replication, which has therapeutic effect, was obtained. After PCR check and sequencing, it was cotransfected to packaging cell 293 LT together with lentiviral packaging plasmids by SSP method, to pack and produce virus. Its titer was assayed. Results PCR and sequence analysis confirmed that MUC16 HS_OVCOV1-LfcinB nuclear nucleotide sequence was correct, lentiviral titer of virus suspension of 1×10~5 U / L. Conclusion The MUC16 HS_OVCOVl-LfcinB gene lentiviral vector was successfully constructed, which laid the foundation for gene therapy and study of anti-ovarian cancer.3 The Effect of MUC16 HS_OVCOVl-LfcinB lentivirus with conditional replication on SKOV3 cells in vitroObjective To investigate the effect of MUC16 HS_OVCOV 1-LfcinB lentivirus with conditional replication on human ovarian cancer cell line SKOV3 in vitro. Methods MUC16 HSOVCOV1-LfcinB lentivirus with conditional replication was transiently transfection SK.OV3 cells through liposome in vitro. After transfection, ovarian cancer cell line SKOV3 cell line were cultured 24 h, 48 h and 72 h, respectively, then their cellular morphology, apoptosis and growth were observed and measured by microscopy, HE and flow cytometry analysis. Results MUC16 HS_OVCOV1-LfcinB lentivirus with conditional replication inhibited cell growth and proliferation, and intruced cell apoptosis. Their apoptosis rates were 6. 57±0. 12,12. 3±0. 45and22. 9±0. 73, respectively, for 24 h, 48 h and 48 h cell culture. Conclusion In vitro, MUC16 HS_OVCOV1-LfcinB lentivirus with conditional replication affected cultured human ovarian cancer cell line SKOV3 and inducted their apoptosis.