Transfected VII<sup>–</sup> virion DNA fosters productive infection.
2017-06-19T17:45:21Z (GMT) by
<p>HeLa cells were cotransfected with purified viral DNAs and an EGFP expression vector. Viral DNAs corresponded to Ad5-WT, Ad5-VII-loxP grown in 293 cells (VII-lox-293), and two independent preparations of Ad5-VII-loxP grown in 293-Cre cells (VII-lox-Cre1 and VII-lox-Cre2). 48 hours after transfection, the cells were harvested and used for the preparation of whole cells protein extracts, total cell DNA, and total cell RNA, as described in Materials and Methods. (A) EGFP Western blot of samples isolated from three independent transfection experiments. Lane 1, untransfected; lanes 2, 6, and 10, cells transfected with Ad5-WT DNA; lanes 3, 7, and 11, cells transfected with VII<sup>+</sup> DNA; lanes 4, 8, and 12, cells transfected with VII<sup>−</sup>DNA preparation 1; lanes 5, 9, and 13, cells transfected with VII<sup>−</sup>DNA preparation 2. (B) Total cellular DNA isolated from these transfections were digested with DpnI to cut transfected, input plasmid DNA, and analyzed by qPCR for Ad5 DNA levels using a primer pair that PCRs across a segment with two DpnI sites. (C) Total cellular RNA was these transfections was analyzed for E1a mRNA levels as described in Fig 5B. n = 3 for (B) and (C) and the values are plotted as mean ± sd.</p>