Dissertation > Excellent graduate degree dissertation topics show

Construction of Human Synuclein-γ Eukaryotic Expression Vector and Its Stable Expression in Glioma Cells

Author: LiangBo
Tutor: ZhangSai
School: Tianjin Medical University
Course: Surgery
Keywords: Synuclein-γ eukaryotic expression vector U373 glioma cell stable transfection real-time RT-PCR flow cytometer
CLC: R739.4
Type: Master's thesis
Year: 2011
Downloads: 57
Quote: 0
Read: Download Dissertation

Abstract


Objective:There is many genes involved in the glioma’s development process. They are interacting through a complex network, genes are the most basic unit of the network. Researching the Glioma on gene level,to further understanding the pathogenesis of glioma and the disease process has a fundamental help.Study a gene’s variated regulation in gliomas has great significance in assessment for the diagnosis, prognosis and therapeutic target of glioma. To establish synuclein-γeukaryotic expression vector,and then use synuclein-y eukaryotic expression vector to construct stably overexpressed SNCG model in human glial tumor U373 cells, and to study how overexpression of SNCG influence on the U373 cell lines from gene,cell cycle and drug intervention three aspects.Methods:First, refer to GenBank database （NM003087）, using Primer 5.0 software design primer SNCG,which is used to be amplified.Subsequently,with a template of plasmid pcDNA3-SNCG,SNCG full length gene was amplified by RT-PCR, of the amplified DNA fragments were purified, the product was purified eukaryotic expression vector pIRES2-EGFP were digested by the target band after electrophoresis, after recovery, with T4 DNA ligase and vector fragment were connected DH5αE. coli with the transformation of the bacteria into uniform coating containing kanamycin 50mg/L of the LB agar plate,37℃inverted culture 12 h, resistant colonies to be grown single colony picked after transfer to liquid medium in shake culture. By plasmid extraction kit instructions mention the small plasmid, recombinant plasmid as a template for PCR amplification of plasmid identification. BamH I and EcoR I and then the double-enzyme digestion cloned into eukaryotic expression vector pIRES2-EGFP.Recombinant bacterium fluid was sequenced by Shanghai Invitrogen Biotechnology Company,and named the correctly recombinant plasmid was pIRES2-EGFP-SNCG.After the identification by restriction endonuclease digestion and the nucleotide sequencing, the recombinant vector was stably transfected into human glial tumor U373 cell lines by G418. The expression of SNCG was detected by real-time RT-PCR and Immunofluorescence,cell cycle distribution was detected by FCM.Result:Verified by RT-PCR and pIRES2-EGFP vector cloning site primers on a bi-directional sequencing, DNA sequencing results confirmed that the recombinant plasmid insert sequence SNCG consistent with GenBank published sequences,It was proved that SNCG recombinant eukaryotic expression vector constructed correctly.The SNCG eukaryotic expression vector was constructed and verified. The U373/SNCG cell line model was established which stably transfected SNCG. After the recombinant plasmid pIRES2-EGFP-SNCG stably transfected U373 cells, used fluorescent microscope observing them in the same field, all the cells showed a visible view in the green fluorescence. The RT-PCR results showed that compared to U373 cells withou transfection,neo mRNA’s expression in U373/neo and U373/SNCG cells were significantly increased; U373/SNCG cells SNCG mRNA expression level was significantly increased, and U373 cells without transfection has no difference between U373/neo cells.The relative fold change of SNCG in U373/SNCG cells was 17 compared with U373/neo cells. Meanwhile, Over-expressing SNCG decreased the blockage of cell mitosis which was induced by anti-microtubule drugs, with decrease in the G2/M phase 17%（P<0.05）.Conclusion:The recombinant expression vector pIRES2-EGFP-SNCG has been constructed and stably transfected in the U373 cell lines successfully, initially confirmed the expression of SNCG had reduced anti-microtubule drug-induced U373 cell mitosis arrest,which lays a base for further study about the role of SNCG in glial tumor’s invasion and metastasis in vitro.

Related Dissertations

  1. Cloning and Expression Analysis of Cold-Related Genes of Ammopiptanthus Nanus,S793.9
  2. Silkworm BmMP54 polyhedrin gene and gene fusion expression and function of the preliminary study,Q78
  3. The Expression and Clinical Significance of Mcl-1 and bar/abl Genes in Chronic Myeloid Leukemia,R733.72
  4. Construction and Identification of PIRES-BMP2-TGFβ3 Bicistronic Eukayotic Expression Vector,R87
  5. Construction of Glycosyltransferase β 3GnT2 、β3GnT8 Eukaryotic Expression Vector and β 3GnT8 RNA Interference Cell Lines SGC-7901 and Effect of β 3GnT8 on the mRNA Expression of Related Genes,Q78
  6. MicroRNA-21 and -141 in patients with epithelial ovarian cancer tissue and serum Expression and Clinical Significance,R737.31
  7. Research of Detection Noroviruses in Different Food Matrices with PGM-MB Technology,TS207.4
  8. Construction of Human KCNJ15 Gene Eukaryotic Expression Vector and in Vitro Investigation of Its Function,R512.6
  9. Researching on Transferring Human Insulin Gene into Chicken,S831
  10. The Construction of Multiple siRNAs Eukaryotic Expression Vector for bcr/abl Fusion Gene,R733.72
  11. Construction of Eukaryotic Expression Vector with GAD67 Gene and Research of Transfecting It into Bone Marrow Stromal Cells,R742.1
  12. Salusin-a eukaryotic expression vector and its expression in HEK293 cells,R541.4
  13. Expression of Bovine Scrapie Prion Protein Gene PrP102-242 and PrP1-264 in COS-7 Cells,S852.659.7
  14. Construction of Eukaryotic Express Vector with Mouse PPARγ Gene and Its Effect on Breast Cancer MDA-MB-231 Cells Cycle,R737.9
  15. Construction of the Expression Vector with a Promoter of Immediate-early Gene (ie1) from White Spot Syndrome Virus and Application of the Vector in Gene Function Analysis,S945.1
  16. Study on Expression and Significance of IFITM1 Gene in the Tissue and Cell of Cervical Cancer,R737.33
  17. Construction of pEGFP-N3-KLF2 Eukaryotic Vector and Impact on LPS-induced TF Expression in THP-1,R363
  18. Construction of Eukaryotic Expression Vector with Bovine Gene Oct4/Sox2/c-Myc/Klf4 and Transfecting Skin Fibroblasts,Q78
  19. The Construction of SOCS3 Eukaryotic Expression Vector and Effection on Apoptosis of the Adipose Cells,Q255
  20. Construction and Expression of Eukaryotic Expression Vectors for Rat Annexin Ⅱ and Tubulin Beta-5 in DRG Cell,R741
  21. Construction of Recombinant Adenovirus Vector with Human SCL Gene,R346

CLC: > Medicine, health > Oncology > Nervous system tumors
© 2012 www.DissertationTopic.Net  Mobile