Construction of the full-length infectious SA<sub>14</sub>-14-2 cDNA as a BAC.
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posted on 2014-07-31, 03:24 authored by Sang-Im Yun, Byung-Hak Song, Jin-Kyoung Kim, Gil-Nam Yun, Eun-Young Lee, Long Li, Richard J. Kuhn, Michael G. Rossmann, John D. Morrey, Young-Min Lee<p>(<b>A</b>) Structure of the SA<sub>14</sub>-14-2 genomic RNA (GenBank accession no. JN604986). NCR, non-coding region; ORF, open reading frame. (<b>B</b>) Diagram of a panel of four overlapping SA<sub>14</sub>-14-2 cDNAs contained in pBAC/Frag-I to IV. SP6 promoter and an artificial run-off site are shown. An asterisk indicates a pre-existing <i>Xba</i>I site at nucleotide 9131 that was inactivated by introducing a silent point mutation, A<sup>9134</sup>→T. (<b>C</b>) Structure of the full-length SA<sub>14</sub>-14-2 cDNA (pBAC/SA<sub>14</sub>-14-2). (<b>D</b>) Functionality of pBAC/SA<sub>14</sub>-14-2. After <i>in vitro</i> transcription with SP6 RNA polymerase, RNA transcripts were electroporated into BHK-21 cells, and infectious plaque centers were determined (RNA infectivity). At 22 and 40 hpt, supernatants from RNA-transfected cells were harvested for virus titration on BHK-21 cells (Virus yield).</p>
