Prnat h1.3 hygro sifluc

The in vitro gene transfer efficiency of CH₁₂K₁₈PEG_5k DNA nanoparticles was evaluated using the reporter plasmid, pRNAT-H1.3/Hygro/siFluc (GenScript, Piscataway, NJ). This plasmid has a coral GFP reporter (cGFP) under CMV promoter control, which can be used to track the transfection efficiency; the plasmid also contains the siRNA construct for firefly luciferase, which uses a CMV-enhanced H1 promoter for siRNA expression. GL261 cells were seeded onto 12-well plates at an initial density of 5.0×10⁴ cells per well and incubated overnight at 37°C. After 24 h, cells were incubated with DNA nanoparticles (2 µg DNA/well) in media for 6 hrs at 37°C. Subsequently, nanoparticles and culture medium were replaced with fresh media. After additional 48 h incubation at 37°C, GFP positive cells were sorted and collected by FACS, after which the GFP positive cell populations were imaged under confocal microscope. Subsequently, 0.25 ml of 1X Reporter Lysis Buffer was added to each wells and then subjected to a freeze-and-thaw cycle. Cells were detached and collected using a cell scraper, and supernatants were obtained by centrifugation. Luciferase activity in the supernatants was analyzed using a standard luciferase assay kit (Promega, Madison, WI) and a 20/20n luminometer (Turner Biosystems, Sunnyvale, CA). Data are shown as relative light unit (RLU)/mg protein.

The plasmid, pRNAT-H1.3/Hygro/siFluc (GenScript, Piscataway, NJ), encoding a GFP reporter gene, siRNA construct for luciferase, and ampicillin resistance was used. This plasmid has a coral GFP marker (cGFP) under CMV promoter control, which can be used to track the transfection efficiency; the plasmid also contains the siRNA construct for firefly luciferase, which uses a CMV-enhanced H1 promoter for siRNA expression. The plasmid were propagated in Escherichia coli DH5α under appropriate antibiotic selection, and collected and purified using EndoFree Plasmid Giga kits (Qiagen Inc., Valencia, CA) per manufacturer’s protocol. Endotoxin levels were < 5 EU/mg plasmid. For flow cytometry analysis of cellular uptake, the plasmid DNA was labeled with Cy5 (Label IT® Nucleic Acid Labeling Kit, Mirus, San Francisco, CA) according to the manufacturer’s protocol.