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 results from flow cytometry analysis are shown above. Percentage of cells expressing GFP is shown on the left, while the percentage of cells with siRNA uptake is shown on the right. Prime-Fect was effective for both pDNA and siRNA delivery in human astrocytes with slightly better performance than the LipofectamineTM 3000 in both cases. Other experimental reagents under development were also promising in some cases.
| Data courtesy of Dr. Xin Liu, Shanghai 9th People’s Hospital (China)|