Dissertation > Excellent graduate degree dissertation topics show

Study on Cloning of Human Telomerase Catalytic Subunit (hTERT) Gene Promoter and Its Activity

Author: WuZuo
Tutor: TanJian
School: Tianjin Medical University
Course: Medical Imaging and Nuclear Medicine
Keywords: human telomerase reverse transcriptase (hTERT) telomerase plasmid gene therapy targeting
CLC: R730.2
Type: Master's thesis
Year: 2011
Downloads: 44
Quote: 0
Read: Download Dissertation

Abstract


ObjectivesWe cloned the hTERT promoter into the pGL3 Vector, resulting in the expression of fluorescent proteins only in telomerase positive cancer cells.MethodsWe uesd RT-PCR and real-time fluorescent quantitative PCR (FQ-PCR, or qPCR) to detect the expression of the hTERT andβ-actin genes in some tumor cell lines and a normal cell line, tested the protein expressing of the hTERT andβ-actin genes by Western-Blotting, amplified and cloned the hTERT core promoter by PCR and inserted into pGL3-Basic vectors, constructed the hTERT promoter recombinant plasmids, detected transcriptional activities of hTERT promoter in cell lines by measuring the luciferase activities.Results1. The RT-PCR showed the expressions of the hTERT mRNA existed in H460, U251, U87, ARO and FRO cells, yet the levels of transcriptional activities were different. But the expression of the hTERT mRNA was do not found in the MRC-5(normal cell). The FQ-PCR showed the expression of the hTERT gene was very high in U251 cells, and the relative concentrations was 0.18% compared withβ-actin (internal reference).2. Western blots demonstrated a positive band at 120 kD, which was the target gene expression of hTERT protein.3. Successfully constructed and identified plasmids pMD-18T and the pGL3 with 3 different length hTERT promoter sequences (260 bp,456 bp and 1453 bp). And successfully constructed and identified plasmid pGL3-hNIS with 260 bp hTERT promoter and hNIS as well.4. Luciferase activity assay showed all three kinds of hTERT promoter fragments could induce the expression of fluorescent proteins, but differences existed in their efficiencies in 5 tumor cells,. The pGL3-204 contained the p260 bp promoter fragment possessed the strongest efficiencies, which could reach 80% of the SV40 promoter, in FRO, H460, U251 and U87cells.ConclusionThe expressions of the hTERT gene was different in tumor cells,and the hTERT promoter could lead the tumor-specific expressions in glioma cells and other tumor cells. The hTERT core promoter had higher promoting efficiency, and could replaced the full length promoter and lead the tumor-specific gene expression.

Related Dissertations

  1. The Expression of Let-7 microRNA during the Process of the Mouse Embryonic Lung Development and the Construction of the Adenovirus Shuttle Plasmid,Q78
  2. Polyethylenimine Conjugated Stearic Acid-g-chitosan Oligosaccharide Micelles for Antitumor Gene Therapy,R450
  3. The Small Organic Molecules Targeted Human Telomeric G-quadruplex DNA: Design, Synthesis and Binding Properties,TQ460.1
  4. The Prokaryotic Expression of Melittin Gene and Its Targeting Transcription in Hela Cells,R346
  5. The Studies to Anti-tumor Effect and Its Mechanisms of Nsaids Combined with Sodium Butyrate on Human Colon Cancer Cell Line HCT-116 in Vitro,R735.35
  6. Clinical Analysis of Multifocal Papillary Thyroid Carcinoma,R736.1
  7. Complex functions drug-loaded microspheres and in spinal cord injury treatment Application of,R943
  8. The Characteristic of the Whole Hepatitis B Virus Gene, Construction of HBV Gene Eukaryotic Cell Expression Vector, and the Efficacy of Adefovir Plus Entecavir or Plus Lamivudine as Rescue Therapies in Chronic Hepatitis B Patients with Entecavir-resistant,R512.62
  9. Experimental Studies on Therapy of Lung Cancer by VEGF-C siRNA Mediated by Liposomes,R734.2
  10. Effect of hTER Site-mutants on Cell Proliferation,Q78
  11. Structure Analysis of the Virulence-related Plasmid from Vibrio Harveyi VIB 645 and Preliminary Study of the PLD Family Protein from Edwardsiella Tarda,S941
  12. Construction of recombinant Clostridium anaerobic eglA p-Her2/neu-IL12 fusion gene expression vector shuttle,R378
  13. Detection of hTERT mRNA and GGT mRNA in the Peripheral Blood of Small HCC Patients and Its Clinical Significance,R735.7
  14. A rocket fast sighting system hardware and software design and analysis,TJ393
  15. The Effect of Total Phenolics from Abnormal Savda Munziq Combined with Chemotherapeutic Agents on HeLa Cells,R737.33
  16. The Significance of P16INK4a,Ki-67 and Telomerase Expression in Squamous Epithelial Lesion of Cervix,R737.33
  17. The Preclinical Study of Imaging and Suicide Gene Therapy of HSV1-tk Reporter Gene to Adenocarcinoma of Lung,R734.2
  18. Therapeutic Effect of Hydrotalcite Combined with Folic Acid on Chronic Atrophic Gastritis Evaluated by Marking Targeting Biopsy,R573.3
  19. Experimental Study of the Effects of Bone Marrow Mesenchymal Stem Cells Transfected with Sonic Hedgehg Gene for the Chronic Ischemic Heart Disease,R541.4
  20. The Clinical Significance of CIN Diagnosis and Follow-Up Visit Caused by Detection of Telomerase in HR-HPV Inflected Patients,R737.33
  21. Adenovirus Delivered Tissue Inhibitor of Metalloproteinases-3 Sensitizes HPV Positive Cervical Cancer Cells to Irradiation in Vitro and Vivo,R737.33

CLC: > Medicine, health > Oncology > General issues > Tumor pathology, etiology
© 2012 www.DissertationTopic.Net  Mobile