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. 2015 Feb 23;10(2):e0118806. doi: 10.1371/journal.pone.0118806

Fig 1. Construction of the Myxoma virus M138L mutant strains.

Fig 1

The M138L Deleted (M138L Del), M138L Revertant (M138L Rev) and M138L STOP strains of MYXV were constructed from the hypervirulent strain Lausanne (WT). A. In the M138L Del strain, most of the M138L coding sequence was replaced by an eGFP-Neo cassette where eGFP is expressed as a fusion protein with the neomycin resistance protein under a synthetic early/late promoter of vaccinia virus (this cassette also contains the LacZ gene). The revertant strain was constructed by replacing the eGFP-Neo cassette in the M138L Del strain by WT sequence. The M138L STOP strain was constructed by introducing a SbfI restriction site in order to create a shift in the reading frame of the M138L gene leading to several STOP codons and to identify this mutant strain. B. Verification of the molecular structure of the mutant viruses by electrophoresis and Southern blotting. Viral DNA was digested with HindIII, resolved by agarose gel electrophoresis, transferred on a nitrocellulose membrane, hybridized with 32P-labeled probes, corresponding to either a fragment of M138L or eGFP and finally submitted to autoradiography. For the M138L probe, the 33,703-bp WT band becomes 22,801-bp for the M138L Del mutant and 33,714-bp for the M138L STOP strain. For the eGFP probe, the band for the M138L Del mutant is 15,242-bp. Marker sizes in Kbp are indicated on the left.