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.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat