Figure 5
The genetic fusion of the inverted Tg-110 and Tg-220 fragments to reconstruct the genomic structure of the MOR42-3 locus. (A) Schematic diagram of fusion of Tg-110-Inverted and Tg-220 fragments. The selection marker cassettes cI-bsr, consisting of the cI and blasticidin S resistance genes (depicted as cB), and cI-spc (depicted as cS) were inserted to the right end of the inverted Tg-110 and to the left end of Tg-220, respectively. 1.3 kb fragments in the left end of Tg-220 and in the right end of Tg-110-Inverted were used as homology arms. Genomic DNA was isolated from the bsr-labeled Tg-110 clone (Tg-110CIBS) and added to the spc-labeled Tg-220 competent cells (Tg-220CISP). The Tg-220 insert was extended to 252 kb by homologous recombination at the overlap region. (B) The fused Tg-250SB insert was evaluated by I-PpoI digestion followed by CHEF. Tg-250SB shows a larger band that corresponds to the fused insert fragment. The closed and open arrowheads represent bands from the 4.2 Mb BGM vector and inserts, respectively. (C) The structure of the Tg-250SB insert was confirmed by Southern blot analysis. Genomic DNA was digested with EcoRI and hybridized with Tg-110 and Tg-220 probes. The genetic fusion of the two inserts is shown as the sum of the respective Tg-250SB bands. The I-PpoI sites are represented by “I”. Lane M, lambda/HindIII fragments or a concatemer of lambda DNA was used as a size marker.