DS
Deborah J. Stumpo
  • Post-transcriptional Gene Expression Group, Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, USA
Research fields
  • Immunology
Mouse Embryonic Fibroblast Cell Culture and Stimulation
Authors:  Lian-Qun Qiu, Wi S. Lai, Deborah J. Stumpo and Perry J. Blackshear, date: 07/05/2016, view: 20833, Q&A: 0
Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to maintain the quiescent status of these cells before they are exposed to pro-inflammatory stimuli for optimal responses. As shown in our previous studies, these mouse fibroblasts do not express Tnf, Csf2 or Il2 mRNAs at levels readily detectable by routine northern blotting techniques (Lai WS et al., 2006).
Measurement of mRNA Decay in Mouse Embryonic Fibroblasts
Authors:  Lian-Qun Qiu, Wi S. Lai, Deborah J. Stumpo and Perry J. Blackshear, date: 07/05/2016, view: 11127, Q&A: 0
mRNA stability control is a critical step in the post-transcriptional regulation of gene expression. Actinomycin D, an antibiotic initially used as an anti-cancer drug, has turned out to be a convenient tool for studying the turnover rates of transcripts in cells, due to its inhibition of mRNA synthesis. Here, we describe a protocol for the measurement of mRNA decay after adding actinomycin D into the medium of stable fibroblast cell lines derived from wild-type and tristetraprolin (TTP)-deficient mouse embryonic fibroblast (MEF) cultures, as well as a protocol for determining the relative transcript abundance using semi-quantitative real-time RT-PCR. Northern blotting or NanoString n-Counter are alternative methods to measure mRNA abundance, which is quantified using a phosphorimager in the former case. This protocol is suitable for studying primary cultured cells and stable cell lines derived from transgenic mice and their respective controls, and provides for direct comparisons of mRNA decay rates in otherwise identical cells with and without the gene of interest.
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