Figure 4

Generation of grlJ^- strains. (A) Targeting vector and recombination strategy. Gene disruption vector for GrlJ: The neomycin resistance cassette (2.2 kb) was obtained from pDNeo2 by EcoRV digestion and cloned into the EcoRV site of the GrlJ gene that was present in the pGEMTeasy vector and the DNA fragment was used for transfection of A×2 cells. (B, C) Confirmation of the recombination event. The recombination event of grlJ^- was analysed by Southern blotting. Transformants were selected with 4 μg/ml G418 for Neomycin resistance. Single colonies were obtained by spreader dilution of the whole pool of transformants onto SM agar plates overlaid with Klebsiella areogenes. Single transformants were then grown with the respective selection medium in a 96-well plate and eventually transferred to a 24-well plate and a 6-well plate. The amoebae were spread on K. aerogenes again and used to isolate genomic DNA for Southern blot analysis. The DNA was digested with either BglII (B) that has recognition sites in the GrlJ gene near the N terminus and 3' of the end of the gene or with EcoRI (C) that has recognition sites outside of the GrlJ gene. Separation of the DNA was in agarose gels (0.7 % agarose). The resulting blot was probed with a C-terminal probe indicated by a bar. WT, A×2; C9 and A4, two independent transformants.