Circadian Bioluminescence Can Be Recorded in Fibroblasts Infected with a Lentiviral Luciferase Expression Vector

<div><p>(A) Circadian reporter constructs used in these studies. Each contains the mouse <i>Bmal1</i> promoter, the firefly luciferase coding region, and the <i>Bmal1</i> 3′UTR, flanked by the long terminal repeats (LTRs) of a lentiviral packaging vector. In (i), a dimerized chick β-globin FII element is inserted between each LTR and adjacent <i>Bmal1</i> sequences. In (ii), a DNA segment composed of the <i>EF1α</i> promoter and a SV40 terminator is inserted between the upstream LTR and <i>Bmal1</i> promoter, and the gfp coding region between the <i>Bmal1</i> UTR and the downstream viral LTR.</p> <p>(B) 3T3 cells were infected with the lentiviral vectors shown above, and equivalent infection levels were verified by real-time PCR to detect integrated viruses. Four days after infection, cells were shocked with dexamethasone to synchronize circadian rhythms, and luciferase output was measured by real-time luminometry.</p> <p>(C) 3T3 cells were infected with different concentrations of the lentiviral reporter vector ii (see [A]), and circadian rhythms were measured as in (B). 10× represents unconcentrated filtered viral supernatant, 1× represents a 10× dilution of this, and 100× was a 10× concentration by ultracentrifugation. The number of viral infection units/plate were approximately 10,000 (1×), 30,000 (3×), 100,000 (10×), 300,000 (30×), and 1,000,000 (100×).</p>