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Retroviral-mediated gene transfer into the WEHI-231 B cell line

University of British Columbia

B lymphocytes play an important role in the immune response against pathogens. Our laboratory is interested in the biochemistry of how antigen engagement of the B cell antigen receptor (BCR) regulates B cell activation and survival. The WEHI-231 murine B cell lymphoma is extensively used as a model system to study this process. WEHI-231 cells undergo growth arrest and apoptosis in response to BCR crosslinking and can be rescued from apoptosis by engagement of CD40. To investigate the signaling pathways that mediate these responses, a common approach is to express altered signaling proteins that may block or activate signaling pathways. We and others have previously used electroporation to express genes of interest in WEHI-231 cells. However, electroporation is an inefficient and timeconsuming method to express genes in these cells. An alternative method to express genes in WEHI-231 cells involves the use of retroviruses as a vehicle for gene transfer. One aim of this thesis was to set up retroviral-mediated gene transfer in our laboratory and optimize retroviral-mediated gene transfer into WEHI-231 cells. By testing different retroviral vectors and methods of infecting WEHI-231 cells, we have optimized this method such that 75% of the cells express the gene of interest after two days. In addition, the remaining non-infected cells can be killed with two additional days of puromycin selection. Thus, using this procedure, one can obtain a pure population of WEHI-231 cells expressing an exogenous gene in four days. In addition, we found that the optimized method of retroviral-mediated gene transfer could be used to express genes in the BAL17 B cell line but not the A20 B cell line. Since sufficient cells for biochemical experiments can now be obtained in four days as opposed to 4-6 weeks for electroporation, the retroviral-mediated gene transfer procedure reported here may expedite the study of signal transduction in B cells. A second aim of this thesis was apply the method of retroviral-mediated gene transfer to manipulate the signal transduction pathway regulated by phosphatidylinositol 3-kinase (PI3K) in WEHI-231 cells. The phospholipid products produced by PI3K regulate many downstream targets. In turn, these targets regulate diverse biological responses such as the prevention of apoptosis, cytoskeletal rearrangements and cell differentiation. PI3K is activated in response to BCR crosslinking, however the downstream targets and biological responses regulated by BCR-activated PI3K remain poorly characterized.

Thesis/Dissertation

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2009-05-25

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http://hdl.handle.net/2429/8142

Microbiology and Immunology

University of British Columbia

UBCV

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