Dissertation > Excellent graduate degree dissertation topics show

Screening Lentiviral Vector with Carrying Effectively siRNA of ROCK2Gene

Author: PengQiang
Tutor: JiangZuo
School: Luzhou Medical College
Course: Surgery
Keywords: SHR ROCK2 RNA interference targets for screening
CLC:
Type: Master's thesis
Year: 2013
Downloads: 3
Quote: 0
Read: Download Dissertation

Abstract


Objective：To screen lentiviral vector carrying siRNA thatcould significantly suppress the ROCK2gene expression in the corpuscavernosum smooth muscle cells of the spontaneously hypertensive rat(SHR). Method:1.We used the organizational adherence method toisolate and culture the SHR corpus cavernosum smooth muscle cells.2.To design the small interference RNA (siRNA) specific to SHRROCK2gene by RNA interfering technique, construct its recombinantlentiviral expression vectors, and identify these vectors by DNAsequencing.①RNAi target sequences were designed, then the targetsequences of Oligo DNA were synthesized and annealed todouble-stranded DNA, which were connected with GP-SupersilencingVector digested by Hpa I、XhoI Short hairpin RNA lentiviral vectors wereconstructed．②The recombinant plasmid was transformed into competentDH5α cells. The positive recombinant colony was selected by ampicillinmedium agar and identified by DNA sequencing.③The recombinedvectors were identified by restriction enzyme analysis and sequencing.Lentivirus was produced after GP-Supersilencing Vector and threepackage plasmids were cotransfected to293T cells.3. Five male SHR cell samples were randomly divided into six groups（each group n=5）,each group for each sample were3×104cells. Group A（control groupwhich untransfected）；Group B（GFP lentiviral transfected group）；GroupC～F（transfected group with lentiviral vector respectively carryingsiRNA1~4targeted to the ROCK2gene）.Transfect SHR corpuscavernosum smooth muscle cells as MOI=80. GFP expression wasobserved under fluorescent microscope after transfection48hours,and themRNA expressions of ROCK2gene in the transfected corpus cavernosumsmooth muscle cells were detected by RT-PCR. Results:1.PCR andDNA sequencing demonstrated that the recombination plasmids targetingthe ROCK2gene were successfully constructed.2.Infection efficiency ofcorpus cavernosum smooth muscle cells of SHR observed underfluorescence microscope was more than50%.3.RT-PCR to detect thetransfected cells ROCK2mRNA expression:The expression of ROCK2mRNA in the corpus cavernosum smooth muscle cells of SHR were verysignificantly decreased in group C、D、F as compared with group A（P<0.01）, resulted in suppression of ROCK2mRNA expression by43.91±8.19℅、47.15±6.64℅、25.7±6.03℅， respectively； and theexpression were significantly decreased in group E as compared withgroup A（P<0.05）, resulted in suppression by16.81±5.94℅. But theexperimental result did not show any significant difference betweengroup B and group A (P＞0.05). Conclusion:1.A lentiviral vector of ROCK2gene RNAi was constructed successsfully by the geneticengineering technology, and it provides a condition forfurther researchED in vitro and vivo of SHR.2.The study has been successfullyconstructed four kinds of lentiviral vector carrying siRNA againstROCK2gene, all of them could suppress the SHR corpus cavernosumsmooth muscle cells ROCK2gene expression, while three kinds oflentiviral vector have stronger inhibition.

Related Dissertations

  1. Expression Dynamics of Pheromone Binding Proteins and Expression Influences by Mating and Knockdown of Cryl in Spodoptera Exigua,S433.4
  2. Molecular Cloning, Mrna Expression and Rnai of Nadph-Cytochrome P450 Reductase Gene in Helicoverpa Armigera (Hǘbner),S435.622.3
  3. Effects of BMPR-IB Gene Silencing by Small Interfering RNA on Apoptosis of Porcine Pollicular Granulosa Cells and Ecpression of BMP Pathy-Way-Ralted Genes,S828
  4. Inhibition of Myostatin (MSTN) Expresstion in Primary Porcine Fetal Fibroblasts by Lentivector-mediated RNAi,S828
  5. A Preliminary Study on the Regulation of Hepatoma Cell Biological Behavior by MCFP,R735.7
  6. The DNA Cloning and RNA Interference of Some Functional Genes in Laodelphax Striatellus (Fallen),S435.112.3
  7. The Effect of RNA Interference-mediated ERCC1 Gene on the Chemo-treatment Sensitivity of NSCLC in Vitro,R734.2
  8. The Influence of RNA Interference Suppression EphB4 Expression for the PANC-1 Cells of Pancreatic Cancer,R735.9
  9. Experimental Study on the Inhibition Effect of siRNA Expressing Vector-based RNA Interference Targeting HBV x in Human Hepatocellular Carcinoma Cell Line HepG2.2.15,R512.62
  10. The Functions of Chitinase Genes from Locusta Migratoria,S433.2
  11. Initial Exploration of the Function of ZBTB8A-a Member of ZBTB Protein Family,Q75
  12. Effects of Lentiviral Vector Mediated RNA Interference of Protein Kinase C Gamma on Morphine Tolerance in Rats,R96
  13. Construction of Silence Vector for Cytochrome b Gene in Nucleus of Arabidopsis Thaliana and the Transformation,Q943
  14. Expression of the POU Domain Transcription Factor Oct4 in Human Transitional Carcinoma Tissue of Bladder and Its Effects on the Biological Behavior of Human Bladder Cancer Cell Line EJ,R737.14
  15. Lisianthus ACC synthase gene RNA interference,S682.19
  16. Preliminary Characterization on Candidate Gene Function of Rice Blast Resistance Gene Pita~2,S511
  17. Constructing and Transforming of Rnai Vectors for 2 osvdac Genes in Rice,S511
  18. Parkinson's disease -related protein Parkin, PINK1 and mortalin interaction studies on mitochondrial homeostasis,R742.5
  19. Study of Transgenic Technology in Processing Tomato Resist Cucumber Mosaic Virus,Q943.2
  20. The Function Characterization of Salt-tolerant Correlative Gene TaSI1 and TaSC in Wheat,Q943
  21. Cellular Model for Inhibition of Grass Carp Reovirus Replication Mediated by RNA Interference,S941

CLC: >
© 2012 www.DissertationTopic.Net  Mobile