Figure 3

Targeted insertion of a reporter gene. (A) In the first step, IRES-tauEGFP was inserted into the targeted site 3 bp downstream of the MOR42-3 stop codon by transformation with a purified DNA fragment consisting of L and R arms (1.0 kb each) homologous to MOR42-3 and the cI-spc selection cassette. In the second step, the selection cassette was removed by transformation with a fragment lacking the selection marker. (B) The targeted insertion of the reporter gene was confirmed by Southern blot analysis using an R homology region as a probe. The genomic DNA of each BGM clone was digested with BamHI and resolved by CHEF, and the changes in the sizes of the BamHI fragments were as expected, indicating the successful targeted insertion of IRES-tauEGFP. The sensitivity to antibiotics is shown. R, resistant; S, sensitive. The BamHI sites are represented by “B”. Lane M, lambda/HindIII fragments were used as a size marker.