Application of the differential display RT-PCR strategy for the identification of inflammation-related mouse genes

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-g. 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.

Identification, structural characterization, and tissue distribution of Tsg-5: a new TNF-stimulated gene

“…52,53 Briefly, total RNA was treated with DNaseI and 200 ng of total RNA were mixed with one of the four anchored oligoT 11 VN primers at 2.5 mM final concentration. Total RNA and anchored oligo(dT) were heated to 701C for 10 min followed by an ice-incubation for 2 min.…”

Exequibilidade do DDRT-PCR para análise da expressão gênica diferencial em células de medula óssea murina

“…3,4 This technique also played an important role in the search for genes involved in regulating embryonic development in rats and in their responses to inflammatory processes. 5,6 In addition, DDRT-PCR also was efficient to identify genes differentially expressed between metastatic tumor and normal prostate cells. 7 Thus, in the present work, we investigated the exequibility of DDRT-PCR in identifying differences in gene expression of bone marrow cells under different conditions (freshly isolated and one week cultivated bone marrow cells).…”