Transfecting Human Astrocytes with RJH Reagents: GFP Expression and siRNA Uptake


Transfecting Human Astrocytes with RJH Reagents: GFP Expression and siRNA Uptake


Astrocytes is an important cell type that participates in hemostasis of brain tissue. They perform several functions, including biochemical support of endothelial cells that form the blood brain barrier, nutrient support to nerve tissues, maintenance of extracellular ion balance, and repair of brain and spinal cord following traumatic injuries. They may also go rouge and give rise to brain tumors (astrocytomas). Modification of astrocytes by genetic means is explored to better understand their physiology and use for therapeutic purposes. They are investigated for spinal cord injuries and modification of cells, either to secrete a desired protein (by an expression vector) or to be transformed into a supportive phenotype (by suppressing certain mediators). It is more desirable to explore non-viral methods to deliver the genetic materials into the cells for safety reasons. Transfection reagents from RJH Biosciences have been explored for this purpose to show that (i) transgenes can be expressed in the cells with a plasmid DNA (pDNA) and (ii) siRNA uptake into the cells is possible.


The primary human astrocytes (Sciencell; Carlsbad, CA) isolated from cerebral cortex were grown in astrocyte complete medium (Sciencell) in multiwall plates 1 day before transfection with complexes. The pDNA contained a GFP expression vector while the siRNA was labeled with FAM. The transfection reagents were Prime-Fect (RJH Biosciences), LipofectamineTM 3000 (ThermoFisher) and other experimental reagents from RJH Biosciences. The complexes were formulated at a reagent:nucleic acid ratio of 5:1 (w/w). After incubating cells with complexes for 1 day (with siRNA) or 2 days (with pDNA), the media was replaced, the cells were washed and recovered (by trypsin) for flow cytometry. The percentage of cells positive for GFP expression or with siRNA uptake are summarized (mean ± SD).


The lanes treated with the scrambled siRNA (#2, 4, 9, 10, and 11) gave band intensities equivalent to the untreated cells (#1), irrespective of the transfection reagent used. With PNKP specific siRNA treatments, significant reductions in the band intensities were evident; LipofectamineTM 2000 at ratio of 1:4 was more potent than 1:2 (siRNA:reagent respectively). All 3 RJH reagents were effective in reducing the PKNP protein levels, indicating the versatility of the reagents. Note that highest silencing was seen with 293-Fect in this study. All 3 RJH reagents, ALL-Fect, 293-Fect and Prime-Fect were effective in silencing the chosen target by 80-90%, based on semi-quantitative densitometric analysis.

| Data courtesy of Dr. Xin Liu, Shanghai 9th People’s Hospital (China)|