Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Construction of AQP1-shRNA Recombinant Adenovirus Expressing Vectors and Identification of Its Function

Author Abdulhakim Abdulmajid Abdulwali Al-Sadhi(HaTiMu)
Tutor LiXiaoMeng
School Northeast Normal University
Course Biochemistry and Molecular Biology
Keywords AQP1 shRNA Adenovirus vector Cell migration
Type Master's thesis
Year 2010
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Aquaporin (AQPs) are the specific the transshipment water channel in the cell membrane. The previous studies showed that AQP1 generally expressed in tumor vascular endothelium, and is also expressed in many other tumor cells. However, whether the expression is an abnormal phenomenon, or whether a key factor in tumor biology, these AQPs expression studies remains unclear. Aquaporin 1 (AQP1) a brief history of research, research can not fully show that the mechanism of action in the body. AQP1 shRNA of adenovirus plasmid constructed in this study, and the use of the AQP1-shRNA adenovirus plasmids interferometer endogenous expression of AQP1 and exogenous expression of AQP1, AQP1 and cell water transporter function and cell movement between relationship. Adenovirus vector system, including the the entry vector pENTR2B purpose vehicle pAd/CMV/V5-DEST first, and the use of recombinant DNA technology, and LR reaction to construct the AQP1-shRNA recombinant adenovirus plasmid, and then double antibiotic selection, different competent cell transformation screening and restriction endonuclease digestion to verify its correctness. Then the correct identification of the molecular level AQP1-shRNA adenoviral plasmid transfection in AQP1 in expression of exogenous CHO cells expressing endogenous AQP1 hepatoma cells SMMC7221 of, AQP1-shRNA experiments by Western blot and immunofluorescence experiments functional identification of adenovirus plasmids, the results show that the the AQP1-shRNA adenovirus plasmids can not only silence the exogenous hAQP1 can also to silence endogenous hAQP1 gene expression, and second plasmid hAQP1-shRNA2 silence the most significant effect. Silence exogenous of AQP1 and endogenous AQP1 expression led him to study the AQP1 and cell water transport function after using the MTT assay membrane water permeability test, scratch test and the Western blot experiments AQP1-shRNA transfection adenovirus plasmids as well as the relationship of cell motility, results AQP1-shRNA adenovirus plasmids either significant interference the exogenous CHO cells AQP1 water permeability, they can interfere in the hepatoma cell SMMC7221 endogenous glycosylated AQP1 water permeability, and AQP1-shRNA adenovirus plasmids silencers AQP1, cell motility by significantly suppressed, while AQP1 repair to involved in cell movement is possible through the reconstruction of the cytoskeleton.

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