Generation of anti-dsDNA antibody production in BALB/c and MRL/MpJ mice injected with plasmid DNA vectors. The three plasmid DNA vectors were used in the experiments shown in (a): pBluescript (pBS, Stratagene, La Jolla, CA, USA; with no eukaryotic elements), pcDNA3E (p3E) and pXAV (containing the CMV promoter myosin light chain 1/3 enhancer +/− the neomycin selection gene respectively25). Endotoxin-free plasmid DNA was prepared from transformed DH5α cells (Life Technologies, Paisley, UK) using a method, which has been described previously.7 Four-week-old female BALB/c and MRL/MpJ mice were obtained from Harlan UK Ltd, Blackthorn, UK; and kept under standard conditions. All procedures were carried out in accordance with Home Office Guidelines. Anaesthesia was induced using fluothane (Rhone Merieux, Harlow, UK) and each group of mice (n = 7) received 25 μl of specific plasmid in saline solution (2 mg/ml) i.m., in the tibilais anterior muscle. In the multiple injection groups this was followed by three further injections at 2-week intervals (at 6, 8 and 10 weeks of age). An additional group of BALB/c mice received multiple intraperitoneal injections of plasmid DNA over the same sequence (a, i.p.). MRL/MpJ mice (n = 7) were injected with pcDNA3E or with saline (b). Two weeks after the final injection, mice were killed, blood immediately removed by cardiac puncture and sera prepared. Serum samples from plasmid-injected BALB/c, MRL/MpJ and MRL/MpJ-Faslpr (positive control) mice were assayed in triplicates for double-stranded DNA antibodies using a Diastat anti-dsDNA kit (Shield Diagnostics, Dundee, UK). Samples were diluted 1 in 100, in PBS containing Triton X-100 (0.01%) and bovine serum albumin (5% w/v) and sodium azide (0.5% w/v). 100 μl of each sample was added to 96-well microtitre plates coated with calf thymus dsDNA, and incubated for 60 min at room temperature. Wells were washed three times in phosphate buffered saline (PBS)/Tween 20 (0.1% w/v), and incubated for 30 min with goat anti-mouse polyvalent immunoglobulin AP conjugate diluted 1 in 3000 in PBS/Tween 20. Samples were washed three times in wash buffer before adding 100 μl of substrate solution (phenolphthalein monophosphate, Mg2+, in buffer solution). Reactions were incubated for 30 min at room temperature, followed by addition of 100 μl of stop solution. Absorbance was measured on a microtitre plate reader at O.D. 550 nm. Significant differences between mean O.D. values were determined using an unpaired t test, with significance set as P ⩽ 0.05.