( 100 g plasmid DNA in 100 l distil...)Detection of Reporter Gene Activity in Cell Cultures and Murine Epidermis After Helios Gene Gun-Mediated Particle Bombardment

100 g plasmid DNA in 100 l distilled H₂O were added. After that, 100 l 1 M CaCl₂ were added dropwise by vortexing the mixture at moderate rate. After incubating the mixture for 10 min at RT, most of the gold and DNA were precipitated. After centrifugation for 15 sec at 14.000 rpm, the pellet was washed three times in 1 ml ethanol (100%, absolute water free). After the final wash, the pellet was resuspended in 200 l ethanol containing 0.1 mg/ml polyvinylpyrrolidone (PVP). To obtain the desired Microcarrier Loading Quantity (MLQ) of 0.5, the final volume was adjusted to 6.0 ml using the ethanol/PVP solution. This suspension was then used for tube preparation which gave a yield of 8090 cartridges containing DNA-coated gold microcarrier with a DLR of 1 g DNA/shot.

CONDITIONS FOR PARTICLE BOMBARDMENT
in vitro experiments. Immediately after aspirating the tissue culture media, confluent HeLa cell monolayers were transfected with single shots of plasmid DNA coated on 0.6 or 1.0 m gold microcarriers. The plastic spacer of the Helios gene gun was placed as close as possible to the target and a helium pressure of 50 psi was applied. After the particle bombardment, 2 ml of media was added and the cell cultures were incubated for 48 hr at 37 C and 5% CO₂.

in vivo experiments. One day prior particle bombardment the abdominal fur of mice was removed by shaving. Murine epidermis was transfected with 1.0 m DNA-coated gold microcarriers by holding the plastic spacer of the Helios gene gun directly against the target site. A helium pressure of 400 psi was applied.

DETECTION OF REPORTER GENE ACTIVITY
Characterization of -galactosidase
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