Optimization experiments were performed in CHO-K1 cells. Cells (2 x 104) were seeded in quadruplicate into 96-well plates and transfected 24 hours later with an in vitro-transcribed CAT-encoding RNA using [A] increasing amounts of RNA with 1.5 µl TransMessenger Reagent and [B] increasing amounts of TransMessenger Reagent with 0.25 µg RNA, as described in the TransMessenger Transfection Reagent Handbook. CAT activity was measured 24 hours post-transfection.
Expression of green fluorescence protein (GFP) in HeLa-S3 cells was examined. Cells (8 x 104) were seeded into 48-well plates and transfected 24 hours later with 0.5 µg of an in vitro-transcribed GFP-encoding RNA (transcribed from P7ASP-GFP/Mlu) using 1 µl Enhancer R and 2.5 µl TransMessenger Reagent. Cells were analyzed 24 hours post-transfection by fluorescence microscopy. Approximately 50% of the cells were successfully transfected. (P7ASP-GFP/Mlu kindly provided by J. Bogenberger, Stanford University Blood Center, Palo Alto, CA, USA.)
[A] Use of TransMessenger Transfection Reagent resulted in an excellent, 60% survival rate post-transfection with beating, functional cardiomyocytes, and a transfection efficiency of 10–13%. [B] High rate of cardiomyocyte and non-cardiomyocyte transfection. Left: Superimposed images showing anti-GFP (green; transfected cell) and anti-α-actinin (red; cardiomyocyte) staining. Right: Anti-α-actinin (cardiomyocyte) staining. Arrows = GFP-positive cardiomyocytes;* = GFP-positive non-cardiomyocytes. [C]Left: GFP-positive cardiomyocytes (green) and non-transfected cardiomyocytes (red; stained with anti-α-actinin) demonstrate equivalent morphologies. Right: Transfected cardiomyocyte and non-cardiomyocyte cells. Data provided by QBMCellScience Inc, Ottawa, ON. Canada