Figure 6

Use of ScIn-2 method for stringent tet-regulated expression of I- Sce I and Rad52. A. Schematic (not to scale) of pIN2-neoMCS. DNA is represented as in Fig 1. MCS, multiple cloning site. B. Schematic (not to scale) of target locus on Rht14-10 after Cre-mediated insertion of pIN2-neoSCE or pIN2-neoR52. C. Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O2 and O5 on cell pellets of three pIN2-neoSCE- and three pIN2-neoR52-transfected Rht14-10 derivatives. Negative (-) and positive (+) controls were, respectively, no cells and cells (TetNeo, see methods) with an integrated tet-regulated neo cassette (expected product: 944 bp). Clones chosen for flp-mediated deletion (10IN-SCE.1 and 10IN-R52.1) are arrowed. D. As in B but after Flp mediated deletion. E. Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O2 and O6 on clones derived from 10IN-SCE.1 (1–12, left) and primers O2 and O7 on clones derived from 10IN-R52.1 (1–12, right). Control reaction contained no cells (-) or genomic DNA generated from pools of flp-treated pIN2-neoSCE/pIN2-neoR52 cells (+). Clones chosen for further analysis (10IN-SCE.1flp1 and 10IN-R52.1flp1) are arrowed. F. Immunoblot analysis of I-Sce I and Rad52 expression in Rht14-10 and, respectively in 10IN-SCE.1flp1 or 10IN-R52.1flp1 at the indicated times after removal or tetracycline.