Rapid and efficient retrovirus-mediated gene transfer into B cell lines.
Murine B cell lines such as WEHI-231, BAL17 and M12.4.1 are frequently used as model systems to study signal transduction, cell cycle regulation, and apoptosis. Dissection of these processes often involves expressing exogenous genes in these cells. Electroporation is an inefficient method to express genes in B cell lines and requires several weeks to isolate and analyze clones, followed by an additional one to two weeks to grow sufficient cells for biochemical experiments (e.g. immunoprecipitations). In this report, we describe an optimized procedure for retroviral-mediated gene transfer into murine B cell lines that allows one to obtain a pure population of cells expressing an exogenous gene within 4 days. Two days post-infection, between 10% (BAL17 and M12.4.1 cells) and 70% (WEHI-231 cells) of the cells express the exogenous gene. Culturing the cells for an additional 48 hours with puromycin kills all the non-infected cells and yields a pure population of cells that express the exogenous gene. Sufficient cells for biochemical experiments can be obtained by expanding the cell culture for an additional 5 to 7 days. This rapid and efficient retroviral-mediated gene transfer procedure can greatly expedite the study of signal transduction and other processes in B cells.
Krebs
D L
DL
The University of British Columbia, Department of Microbiology and Immunology, Vancouver, Canada.
Yang
Y
Y
Dang
M
M
Haussmann
J
J
Gold
M R
MR
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D017452:Receptors, IgG / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics*; D013997:Time Factors
D008164:Luminescent Proteins; D017452:Receptors, IgG; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins