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Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes
A M Silva1, E G Pires, E F Abrantes
1Laboratório de Inflamação, Instituto Ludwig de Pesquisa sobre o Câncer, São Paulo, Brasil.
Abstract:
The inflammatory response elicited by various stimuli such as microbial products or cytokines is determined by differences in the pattern of cellular gene expression. We have used the differential display RT-PCR (DDRT-PCR) strategy to identify mRNAs that are differentially expressed in various murine cell types stimulated with pro-inflammatory cytokines, microbial products or anti-inflammatory drugs. Mouse embryonic fibroblasts (MEFs) were treated with IFNs, TNF, or sodium salicylate. Also, peritoneal macrophages from C3H/Hej mice were stimulated with T. cruzi-derived GPI-mucin and/or IFN-gamma. After DDRT-PCR, various cDNA fragments that were differentially represented on the sequencing gel were recovered, cloned and sequenced. Here, we describe a summary of several experiments and show that, when 16 of a total of 28 recovered fragments were tested for differential expression, 5 (31%) were found to represent mRNAs whose steady-state levels are indeed modulated by the original stimuli. Some of the identified cDNAs encode for known proteins that were not previously associated with the inflammatory process triggered by the original stimuli. Other cDNA fragments (8 of 21 sequences, or 38%) showed no significant homology with known sequences and represent new mouse genes whose characterization might contribute to our understanding of inflammation. In conclusion, DDRT-PCR has proven to be a potent technology that will allow us to identify genes that are differentially expressed when cells are subjected to changes in culture conditions or isolated from different organs.
Insights
Differential display RT-PCR (DDRT-PCR) identified novel genes involved in cellular inflammation. This method effectively reveals changes in messenger RNA (mRNA) expression in response to various stimuli, advancing our understanding of inflammatory processes.
Area of Science:
- Molecular Biology
- Immunology
- Genomics
Background:
- Cellular gene expression patterns dictate inflammatory responses to diverse stimuli like microbial products and cytokines.
- Understanding these expression patterns is crucial for deciphering the mechanisms of inflammation.
Purpose of the Study:
- To identify novel messenger RNAs (mRNAs) with differential expression in murine cells stimulated with pro-inflammatory agents.
- To explore the utility of differential display RT-PCR (DDRT-PCR) in discovering inflammation-related genes.
Main Methods:
- Utilized differential display RT-PCR (DDRT-PCR) on mouse embryonic fibroblasts (MEFs) and peritoneal macrophages.
- Stimuli included interferons (IFNs), tumor necrosis factor (TNF), sodium salicylate, and T. cruzi-derived GPI-mucin with IFN-gamma.
- Recovered, cloned, and sequenced differentially expressed cDNA fragments.
Main Results:
- Out of 28 recovered cDNA fragments, 16 were tested, and 5 (31%) showed modulated mRNA levels in response to stimuli.
- Identified known proteins not previously linked to the specific inflammatory triggers used.
- 8 of 21 sequenced fragments (38%) represented novel mouse genes with potential roles in inflammation.
Conclusions:
- DDRT-PCR is a powerful technique for identifying differentially expressed genes under various cellular conditions.
- This approach facilitates the discovery of new genes contributing to the understanding of inflammatory pathways.
- The study highlights previously unrecognized molecular players in cytokine and microbial product-induced inflammation.