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.
Regulation of the immune response at the molecular level requires the closely controlled expression of genes, some of which must be silenced, while others must either be expressed constitutively or only in response to specific stimuli. Thus, proper regulation of gene expression, particularly cytokine secretion, is essential to assure that protective immunity is generated and maintained and inflammatory and autoimmune disorders are avoided. The expression of cytokine genes by T cells is largely governed by exogenous factors. These factors can lead to the rapid and transient induction of cytokines, influence the subset of cytokines a T cell is capable of expressing, or both. These effects are mediated by transcription factors that bind to the promoter and to additional regulatory elements, including enhancers, silencers and boundary elements, which may lie nearby or 50 kb or more upstream or downstream of the gene they help to regulate1. The identification of cytokine gene regulatory elements, the transcription factors that bind to them, and the epigenetic processes and modifications that modulate transcription factor binding and thereby influence cell fate and function, are an important area of contemporary immunological investigation2.
Nowadays, discovery of distal regulatory elements typically begins with the use of complementary bioinformatic and epigenetic analyses to detect evolutionarily conserved sequences and chromatin and DNA modifications, respectively, that are commonly associated with regulatory elements3. These candidate regions must then be evaluated to determine if they do regulate gene transcription, and, if so, if their function is affected by or dependent on specific stimuli and transcription factors. Whereas certain functions of transcriptional regulatory elements can only be detected in situ or in stably transfected or transduced cells, classical enhancer activity can be detected through the use of reporter constructs that are transiently transfected into appropriate cell types. Long-term human or mouse T cell lines are commonly employed for this purpose, because they can be easily, rapidly and reproducibly transfected and studied. It is also possible to determine the effects of specific transcription factors on enhancer activity by co-transfection of reporter constructs with constructs directing the constitutive expression of signaling molecules or transcription factors not present in that cell line. However, receptors involved in T cell activation and the signaling pathways and transcription factors downstream of these receptors may not be expressed or act in these long-term cell lines in a manner that is truly representative of primary T cells. Self-inactivating retroviral-based or lentiviral-based systems have been used to introduce reporter constructs that become stably integrated into the genome of primary mouse and human T cells4. However, such constructs may be subjected to integration site effects that can mask or modify the enhancer function or lead to confusion as to whether the effect observed reflects enhancer, silencer or boundary element activity. Furthermore, the development of vectors and production of retroviral stocks can be laborious and poses biohazardous risks.
The recent introduction of Amaxa® nucleofection technology has allowed efficient and rapid gene transfer into resting and activated primary human and mouse T cells5,6. In this study, we describe an optimized nucleofection procedure for the transient transfection of luciferase reporter constructs driven by regulatory elements from the murine interferon-γ (Ifng) locus into primary mouse CD4+ TH0, TH1 and TH2 and CD8+ effector T cells and for determination of their effects on expression following stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies, IL-12 plus IL-18 or the combination of these stimuli. This method should be applicable or adaptable to studies of transcriptional or translational regulation, transcription factor function and signaling in primary murine T cells.