Electroporation is one of the most common methods used transform mammalian cells with plasmids. This method is versatile and can be adapted to meet the requirements of many cell lines. However, sometimes the efficiency of this method is low. We demonstrate that dimethyl sulfoxide (DMSO) facilitated a better DNA uptake in four different cell lines (HL60, TR146, Cos-7 and L132). The cells were electroporated with a beta-Gal expression plasmid in a medium containing DMSO (1.25%) during, and for 24 h after the pulse. In all these cells a dramatic (up to 8-fold) increase in transfection efficiency occurred after this treatment. This method opens up the possibility of using electroporation even in cells which are difficult to transfect.
The S100 proteins MRP8 and MRP14 have been shown to be expressed by myeloid cells during inflammatory reactions. Since the majority of S100 proteins exhibit their biological activity when associated as complex it was investigated whether murine MRP8 and MRP14 form heterodimers and whether this complex may bind lipids of the cell membrane. This is of particular importance since their anchoring into the plasma membrane is unclear although upon calcium binding the proteins translocate from the cytoplasma to the cytoskeleton and the plasma membrane. Using recombinant proteins we could show that not the monomers but only the heterodimers specifically bind arachidonic acid. This finding opens new perspectives for the role of MRP8 and MRP14 in acute and chronic inflammatory processes.
S100A9, also referred to as MRP14, is a calcium-binding protein whose expression is tightly regulated during differentiation of myeloid cells. The present study was performed to study the cell type-and differentiationspecific transcriptional regulation of the S100A9 gene. Analysis of the S100A9 promoter in MonoMac-6 cells revealed evidence for a novel regulatory region from position ؊400 to ؊374 bp, termed myeloid-related protein regulatory element (MRE). MRE deletion resulted in a 5.2-fold reduction of promoter activity. By electrophoretic mobility shift analysis two nuclear complexes binding to this region were identified and referred to as MRE-binding complex A (MbcA) and MRE-binding complex B (MbcB). By mutagenesis the MRE-binding motif could be narrowed to a 12-bp region. The relevance of MRE is deduced from the observations that the formation of either MRE-binding complex A or MRE-binding complex B strongly correlated with S100A9 gene expression in a cell type-specific, activation-and differentiationdependent manner. Moreover, DNA affinity chromatography and Western blot studies indicate that a Kruppelrelated zinc finger protein and the transcriptional intermediary factor 1 (TIF1) are involved in an MREbinding complex, thereby regulating the S100A9 gene expression.
Systemic lupus erythematosus T cells display decreased amounts of TCR ζ mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR ζ promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR ζ promoter, centered on the −390 nucleotide. Transfection of T cells with an antisense CREM α plasmid reduced the binding of CREM to the TCR ζ promoter, as shown by chromatin and reporter chromatin immunoprecipitation assays, and enhanced the production of TCR ζ mRNA and protein. Mutagenesis of the −390 CRE site prevented the binding of CREM to the TCR ζ promoter. The mechanism of CREM-mediated repression appears to be chromatin dependent, because antisense CREM promotes the acetylation of histones on the TCR ζ promoter. Finally, we established an enhanced binding of CREM to the TCR ζ-chain promoter in systemic lupus erythematosus cells compared with control T cells. Our studies demonstrate that CREM α binds to the TCR ζ promoter and repress its activity.
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