Preparation of DNA-Coated Gold Microcarriers
On the day prior to vaccination, pCMV-S plasmid DNA was precipitated onto gold microcarriers as detailed in the Helios gene gun system instruction manual. Briefly, 8.3 mg of 1 m gold microcarriers was resuspended by sonication in 100 l of 0.05 M spermidine. Eighteen micrograms (18 g) of DNA at a concentration of 1 mg/ml in endotoxin-free water was then added and sonicated; 100 l of 1 M CaCl2 was then added dropwise. This gold-DNA mixture was allowed to stand for 10 min before being washed 3 times in 250 l of 100% ethanol. After the final wash, the pellet was resuspended in 200 l of 0.025 mg/ml polyvinylpyrrolidone (PVP) in 100% ethanol, transferred to a 15 ml tube, and made up to 1 ml with PVP/ethanol. This resulted in a microcarrier loading quantity (MLQ) of 0.5 mg of gold per shot and a DNA loading ratio (DLR) of 2 g/mg gold, which results in the delivery of 1 g of DNA per shot.
Loading DNA/Microcarrier Suspension into GoldCoat Tubing
This was performed as detailed in the Helios gene gun system instruction manual, with 1 ml of DNA/microcarrier suspension being used to produce 17 coated 0.5-inch cartridges, which were then stored overnight at 4C with desiccant prior to use.
Vaccination of Mice
Female Balb/c strain mice aged 68 weeks were obtained from Charles River, UK, and housed at the Biomedical Services Unit, University of Nottingham, UK.
For gene gun delivery, the abdominal fur of each mouse was removed with
electric clippers prior to each vaccination. The barrel liner of the Helios
gene gun was then held directly against