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. 2013 Jul 4;9(7):e1003465. doi: 10.1371/journal.ppat.1003465

Figure 1. Construction of recombinant MYXVs.

Figure 1

A) Construction of vMyx-M029KO virus. The M029 coding sequence was replaced by a DNA cassette where eGFP is expressed under a poxvirus synthetic early/late promoter (sE/L). The final recombination plasmid has 5′ partial M028L sequence, eGFP and M030L sequence and was constructed by three-element recombination of the Multisite Gateway system. The revertant virus vMyx-M029KO-Rev was constructed by replacing the eGFP cassette in the knockout virus with a plasmid having M028-M029-M030 gene sequence. B) Construction of vMyx-M029ID virus. A DNA cassette where eGFP expressed under a poxvirus synthetic early/late promoter (sE/L) was inserted within M029 gene for disruption of M029 ORF. The final recombination plasmid has 5′ partial M028L sequence, 5′ partial M029L sequence, eGFP, 3′ partial M029L sequence and M030L sequence and was constructed by three-element recombination of the Multisite Gateway system. The revertant virus vMyx-M029ID-Rev was constructed by replacing the eGFP cassette with a plasmid having the intact M028-M029-M030 gene sequence. C) Confirmation of purified vMyx-M029KO and vMyx-M029ID viruses. PCR using primers combination of 3 and 4 (top panel) and 1 and 2 (bottom panel) used for viral DNA from vMyx-M029KO (lanes 2 and 3), vMyx-M029ID (lanes 4 and 5), vMyx-Lau (lane 6). Lane 1 represents known size DNA ladder.