Figure 4
Cre-mediated insertion by ScIn-2. A. Schematic (not to scale) of the target locus in clone Rht14-19 or Rht14-10 before (a) and after (b) Cre-mediated insertion of pIN-2, and after flp-mediate deletion (c). DNA is represented as in Fig. 1 with recognition sites for Hind III (H) and Bgl II (B) and PCR primers indicated. B. Ethidium bromide stained agarose gel of electrophoresed PCR products generated with the primers O1 and O3 and cell pellets of 9 MPA/Xr, GFP-negative clones selected after transfection of Rht14-10 (Rht14-10IN) or of Rht14-19 (Rht14-19IN) with pIN-2 and pMC-Cre. M = marker DNA. Negative (-) and positive (+) controls used, respectively, no DNA and pTIGHTgpt DNA (expected product: 555 bp). C. Southern blots of genomic DNA isolated from Rht14-10 and derivatives and digested with Bgl II (left), or from Rht14-19 and derivatives and digested with Hind III (right). Derivatives before (10IN5 and 19IN3, 4 and 5) and after (10flp9.2 and 3 and 19flp9.2 and 12.3) Flp-mediated deletion are analysed. The probe was a fragment of the d2EGFP gene (methods).