Luciferase Assay

Rats were euthanized 4 hr after the injection of pCMV-Luc plasmid DNA, and tissue samples were collected from liver, pancreas, and other organs as previously described21, 22, 23 and kept at −80°C until use. Lysis buffer (2 mL) (0.1 M Tris-HCL, 2 mM EDTA, and 0.1% Triton X-100 [pH 7.8]) was added to each sample (∼200 mg wet tissue), and the tissue samples were homogenized for 30 s with the tissue homogenizer (ULTRA-TURRAX T25 digital, IKA, Staufen, Germany) at maximum speed. The tissue homogenates were centrifuged in a microcentrifuge for 10 min at 13,000 × g at 4°C. The protein concentration of the supernatant was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) based on Coomassie blue assay strategy. Supernatant (10 μL) was mixed with luciferase assay reagent (100 μL), and the luciferase activity was measured in a luminometer (Luminescencer Octa AB-2270, ATTO, Bunkyo-ku, Tokyo, Japan) for 10 s. The luciferase activity is presented as relative light units per mg of protein.