Authors
Jamie Schoenborn, Masayuki Sekimata, William Weaver, and Christopher Wilson
Summary
Transient transfection is a useful procedure by which to address the function of regulatory elements and transcription factors involved in gene regulation. However, primary mouse T cells are difficult to transfect. This protocol describes optimized conditions for transient transfection of primary mouse T cells with luciferase reporter constructs under the control of regulatory elements from the murine Ifng locus using the Amaxa Nucleofector ®. Primary CD8+ and CD4+ TH0, TH1, and TH2 effector T cells were generated in vitro, then transfected with pGL-Firefly luciferase constructs under the control of the Ifng promoter and candidate regulatory elements along with a thymidine kinase- luciferase transfection control. Following a brief rest period, cells were stimulated with anti-CD3 plus anti-CD28 antibodies, IL-12 plus IL-18, or a combination of these stimuli and then were processed for dual luciferase reporter assays. While this protocol was designed to evaluate the effects of distal regulatory elements on Ifng expression, it could be adapted for use in other gene expression, signaling, and biochemical analyses of primary mouse T cell function.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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