Transfection of bone marrow-derived mast cells for transcription factor luciferase reporter assays
Related Journal & Article Information
Manuscript:
Journal: Nature Immunology
Article Title: Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1
Introduction
Mast cells are now being understood as important intitiators and regulators in both innate and adaptive immunity in addition to their traditional roles in allergic and IgE-mediated immune responses.Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for example, transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells as well as the ability to over-express or reconstitute wild-type and/or mutated constructs into wild-type or knockout cells for analysis of functional responses such as cytokine production, apoptosis, migration etc.
Materials
Reagents
Amaxa solution T, interleukin 3,
Equipment
Amaxa Nucelofector, beta scintillator counter
Procedure
1. Maturate mast cells from bone marrow using interleukin 3. Isolate bone from femurs and tibias of 8-12 Pac-1-/- and Pac-1+/+ week old mice. Grow cells at 4×105 cells/mL in RPMI 1640 medium (Life Technologies, Rockville) supplemented with 10% fetal bovine serum (HyClone, Logan), 100 μg each of penicillin and streptomycin per mL, 2mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES and 5 ng/mL of recombinant murine IL-3 (Peprotech) for 4-6 weeks at 37oC in 5% CO2. By 4-6 weeks in culture, greater than 98% of cells should be c-kit and FcεRI positive as assessed by fluorescein isothiocyanate (FITC)-labelled anti-c-kit (BD, Pharmingen) and anti-DNP IgE (Sigma) antibodies respectively using Flow Cytometry. Cell morphology should also be assessed by cytospin and staining with giemsa and toluidine blue. To stimulate, sensitise cells with anti-DNP IgE mAb (100 ng/mL, Sigma) for 18 hours, wash twice to remove unbound IgE and stimulate with 20 ng/mL of DNP-HSA (Sigma) for chosen times.
2. For the measurement of transcriptional Elk-1 activity, transfect 4 × 106 BMMCs with 1.75 μg pG5E4D38–Luc, 1.75 μg pSG-Gal-Elk-1 (both plasmids kindly provided by Dr. Peter Shaw, University of Nottingham, UK, Frost et al, 1997) and 1 μg of the lacZ-expression vector pCMVβ (Clonetech, to allow standardization of the measured luciferase activity) using solution T and an Amaxa nucleofector device (Köln, Germany; set to program T-016). In addition, “mock-transfect”cells with PBS in order to measure the background luciferase and β-galactosidase activities. This Elk-1 reporter system is based on a hybrid fusion protein consisting of the DNA binding domain of the yeast transcription factor Gal4 and the transactivation domain of Elk-1 that binds via its Gal4 moiety to the pG5E4D38–Luc plasmid and drives the transcription of the luciferase gene only if it is phosphorylated by ERK within its Elk-1 moiety (Kortenjann et al, 1994).
3. For the measurement of transcriptional NFAT:AP-1 activity, transfect BMMCs with 1 μg pCMVβ and 3.5 μg of the NFAT reporter plasmid pNFAT-Luc (kindly provided by Dr. Gerald Crabtree, Stanford University, Stanford, USA) as described above. This NFAT reporter carries three tandem copies of the distal NFAT:AP-1 composite element of the human IL-2 promotor (Durand et al, 1988), which were cloned upstream of the minimal IL-2 promotor (from position -89 to +51) and the luciferase reporter gene. Consequently, this reporter is only transactivated when both NFAT and AP-1 are present in the nucleus and assembled to a ternary complex (Fiering et al, 1990; Macian et al., 2001). This widely used reporter plasmid responds to all known NFAT family members and can be activated in a NFAT-dependent manner in a variety of cell types.
4.Following transfection with reporter plasmids, plate BMMCs in 3 mL culture medium (RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 μg each of penicillin and streptomycin per mL, 2mM L-glutamine (Life Technologies), 50 μM β-mercaptoethanol, 1% HEPES and 5 ng/mL of recombinant murine IL-3 (Peprotech)) and allow to recover for 24 h. Then sensitise with 100 ng/mL anti-DNP IgE antibodies for 18 hours. Subsequently, split the cells and either stimulate with 20 ng/mL DNP-HSA for 6 h (Elk-1) or 8 h (NFAT:AP-1) or leave untreated. Following stimulation, lyse the cells in luciferase lysis buffer (Promega) and determine the raw luciferase activities using a luciferase activity kit (Promega). As an internal control for transfection efficiency, measure the β-galactosidase (β-gal) activities using Galactostar and Galacton reagents (Tropix). The background β-gal activity of mock-transfected cells should be subtracted from the lysates of transfected BMMCs and the relative β-gal activity of each sample was calculated. In order to obtain the standardised luciferase activity, divide the luciferase activity of each sample by the corresponding relative β-gal activity.
5. For over-expression or rescue experiments, transfect mast cells with 2ug of WT Pac-1, C257S Pac-1 or pMIG vector control.
Troubleshooting
Critical Steps
Anticipated Results
References
Jeffrey, K. L., Brummer, T., Rolph, M. S., Liu, S. M., Callejas, N. A., Grumont, R. J., Gillieron, C., Mackay, F., Grey, S., Camps, M., Rommel, C., Gerondakis, S. D., Mackay, C. R. Positive regulation of immune cell function and inflammatory responses by phosphatase PAC-1. Nat Immunol 7, 274-83 (2006).
Frost, J. A., Steen, H., Shapiro, P., Lewis, T., Ahn, N., Shaw, P. E., Cobb, M. H. Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins. Embo J 16, 6426-38 (1997).
Kortenjann, M., Thomae, O. & Shaw, P. E. Inhibition of v-raf-dependent c-fos expression and transformation by a kinase-defective mutant of the mitogen-activated protein kinase Erk2. Mol Cell Biol 14, 4815-24 (1994).
Durand, D. B. et al. Characterization of antigen receptor response elements within the interleukin-2 enhancer. Mol Cell Biol 8, 1715-24 (1988).
Fiering, S. et al. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev 4, 1823-34 (1990).
Macian, F., Lopez-Rodriguez, C. & Rao, A. Partners in transcription: NFAT and AP-1. Oncogene 20, 2476-89 (2001).
Acknowledgements
Keywords
mast cells, BMMC, transfection, nucleofector, amaxa, transcription factors, luciferase reporters
Figure 1
Reduced transcriptional Elk-1 and NFAT:AP-1 activity in Pac1 minus/minus BMMCs. Re-expression of Pac1 but not Pac1C257S could enhance Elk-1 activity in Pac1 minus/minus BMMCs.
a, Reduced transcriptional Elk-1 and b, NFAT:AP-1 activity in Pac1 minus/minus BMMCs as measured by standardized luciferase activity of Gal-Elk-1 or NFAT:AP-1 reporter in Pac1+/+ and Pac1 minus/minus BMMCs transfected using Amaxa Nucleofector and activated for 6 h (Elk-1) or 8 h (NFAT:AP-1) with Fc_RI crosslinking. RLU is relative luciferase activity. c, (Re)introduction of Pac1 but not Pac1C257S into Pac1 minus/minus BMMCs increases Elk-1 activity. BMMCs were co-transfected with Gal-Elk-1 reporter constructs and empty vector alone, full length WT Pac1 or Pac1C257S. Error bars represent standard deviation of triplicate samples. Similar results were obtained in 5 independent experiments.
