Erythroleukemia cell lines (MEL, SKT6 and Rauscher cells) stable transfection with DNA minigene using DreamFect Gold

Proliferative erythroleukemia cells (MEL, STK6 and Rauscher cells) were stably transfected with 0.5 to 1 μg plasmid DNA (minigene) using DreamFect Gold reagent, in 24-well tissue culture plates.

This article demonstrates that due to its low toxicity level and to its high transfection capacities, DreamFect Gold from OZ Biosciences is the reagent of choice for stably transfecting erythroleukemia cell lines (MEL, STK6 and Rauscher cells).

article reference: Cell Signal. 2013 Aug 30. pii: S0898-6568(13)00246-5. 

Combined inhibition of PI3K and activation of MAPK p38 signaling pathways trigger erythroid alternative splicing switch of 4.1R pre-mRNA in DMSO-induced erythroleukemia cells.

Breig O, Theoleyre-Schaal O, Baklouti F.

AbstractThere is increasing evidence showing that many extracellular cues modulate pre-mRNA alternative splicing, through different signaling pathways. We here show that 4.1R exon 16 splicing is altered in response to specific signals. The switch from erythroblastic isoform lacking exon 16 to mature erythrocytic isoform containing this exon is tightly regulated during late erythroid differentiation, and blocage of this splicing switch in erythroleukemia cells is seen as a consequence of the deregulation of important regulatory pathways. We support that combined inhibition of PI3K and activation of p38 signaling pathways impinge on erythroid 4.1R pre-mRNA alternative splicing switch, and on cell differentiation as witnessed by hemoglobin production. By contrast, MEK/ERK signaling appeared not to affect neither cell hemoglobin production nor erythroid 4.1R pre-mRNA splicing. We also found that the signal-induced alternative splicing is not typically distinctive of EPO-non-responsive cells, but operates in EPO-responsive cells as well. Pre-mRNA splicing is a major regulatory mechanism at the crossroad between transcription and translation. We here provide evidence that inhibition of PI3K activates the splicing switch in a promoter-dependent manner, whereas p38 activation induces this event in a promoter-independent fashion. Our data further support that contitutive activation of EPO-R by the viral protein gp55 and the short form of the tyrosine kinase receptor Stk, transduces PI3K proliferation signal, but not MAPK p38 differentiation signal. Concurrently, this work lend credence to the concept that DMSO triggers transient activation of p38 signaling and irreversible inhibition of PI3K/AKT signaling pathway, hence uncovering an old conundrum regarding the mechanism by which DMSO induces erythroleukemia cell differentiation.

DreamFect™Gold allows transfecting all types of nucleic acids with a very high efficiency. Due to its formulation, DreamFect™Gold delivers a large quantity of nucleic acids leading to higher protein expression compared to other transfection reagents. It is fully biodegradable and does not interfere with cellular mechanisms.