Quantitative Genomic PCR Estimate of Integrated Viral Trangenes per Genome

<div><p>(A) Diagram of integrated viral transgene and location of PCR primers spanning the IRES-eGFP junction that were used to amplify the transgene.</p> <p>(B) Ethidium-bromide-stained gel showing 638-bp product (indicated by arrow) amplified from the experimental and standard reactions. Lanes 1–6 contain samples amplified from the six treated animals exhibiting restored sight. No product was observed in untreated GUCY1*B (lanes 7 and 8) or wild-type (lane 9) chickens. A blank water control is shown in lane 10. Lanes 11–14 contain product amplified from 300,000, 150,000, 30,000, and 15,000 copies of the transducing vector. DNA size ladders, 1 kb and 123 bp, were run to the left of the samples and standards.</p> <p>(C) Plot of amount of product obtained from the standard PCR reactions (black) best fit with a sigmoid curve ( <i>f</i> = <i>y</i>₀ + <i>a</i>/(1 + exp(−( <i>x −x</i>₀)/ <i>b</i>)) <i>^c</i> with <i>r</i>² = 0.999 . The products obtained from each experimental sample (red) fell within the linear portion of the amplification curve, and the number of transgenes present in each sample was calculated using the equation for the best-fit curve. Individual values plotted are mean ± SEM ( <i>n</i> = 3). </p> <p>(D) Comparisons of integrated viral transgenes per genome, dark-adapted ERG b-wave amplitudes (1 Hz), volitional behavior scores, and optokinetic reflex scores. The data for the ERG and behavioral tests were collected at the end of the 6-wk study period. The results for the six treated animals exhibiting sight and for age-matched untreated GUCY1*B and wild-type RIR animals are shown. NA, not assayed; ND, not detected.</p>