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The Construction of Survivin shRNA Lentiviral Vector

Author: YuFeng
Tutor: LiuJing
School: Central South University
Course: Internal Medicine
Keywords: Lentiviral vectors Multiple Myeloma RNA interference
CLC: R733.3
Type: Master's thesis
Year: 2011
Downloads: 64
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Abstract


Background and Purpose: Multiple Myeloma (Multiple Myeloma, MM) is a malignant clonal plasma cell proliferative diseases, occur in the elderly. Although there are several joint program of chemotherapy and hematopoietic stem cell transplantation applications, MM is still a poor prognosis of the disease. In recent years, with the in-depth study of pathogenesis of MM, apoptosis and anti-apoptotic regulation disorder is one of the main pathogenesis of MM, studies confirm PDCD5 myeloma protein can promote apoptosis, decreased survivin protein expression, enhanced caspase -3 (?) Tongue sex. Survivin is a member of the IAP family, can inhibit caspase-3 activity. But PDCD5 enhanced caspase-3 (?) Mainly through the tongue of whether downregulation of survivin protein achieved remains unclear. To further investigate whether survivin PDCD5 enhanced caspase-3 (?) Tongue of apoptotic pathway important target molecules, we need to use RNA interference to inhibit multiple myeloma cells survivin gene expression, observe whether the enhanced influence PDCD5 Effects of caspase-3 activity. Because multiple myeloma cell line structure dispersed in non-dividing phase, liposomes, retroviral vector (MLV) transfection inefficient, lentiviral vectors can improve transfection efficiency and to achieve stable gene expression, 'We Construction of lentiviral vectors achieved by RNA interference silencing of survivin gene expression. Methods: Literature design for effective survivin gene expression shRNA sequences, chemical synthesis survivin shRNA primers using gene recombination technology to connect to pRNAT-U6.2/lenti plasmid, using BamH1, Xho1 double digestion electrophoresis, DNA sequencing recombinant clones. Expanding culture positive clones, plasmid amount extraction. Recombinant cloning vector and helper plasmids were transfected by LipofectamineTM2000 HEK293FT cell supernatants were collected, centrifuged to collect the virus particles, stored at -70 ℃. HEK293T cells infected with lentivirus fluorescence microscopy of GFP expression. Results: confirmed by double digestion of pRNAT-U6.2/Lenti shRNA lentiviral expression vector fragment is inserted correctly, DNA sequencing of the inserted fragment confirmed the correct sequence, the cotransfected 293FT cells and 293T cells infected with lentivirus, fluorescence microscopy visible GFP protein. Conclusion: We successfully constructed human survivin gene shRNA 'lentiviral vector for RNA interference technology to further study whether survivin PDCD5 enhanced caspase-3 activity in apoptotic pathways provide the basis for an important target molecules.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Bone marrow tumor
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