BAC Modification through Serial or Simultaneous Use of CRE/Lox Technology
Figure 6
Analyses of simultaneous deletions. (a) The BAC construct with targeted insertions in Hoxb1 and Hoxb2 used as a substrate for assaying Cre-mediated recombination (top). This construct contains one LoxP-flanked Kanamycin cassette and one Lox5171-flanked Ampicillin cassette. Successful recombination (middle) will eliminate the Kanamycin and Ampicillin resistance cassettes and leave a LR-LoxP and LR-Lox5171 recombination resistant site at the respective locations. Recombination between mismatched sites (LoxP × Lox5171) (bottom) will result in the deletion of both cassettes and leave a single hybrid Lox site between segments of the Hoxb2 and Hoxb1 genes. The symbols for each of the components in the diagrams are noted below. (b) NheI digest of clones resulting from Cre-mediated recombination of the BAC substrate. Green arrows indicate features of a successful/specific multiple recombination. Black arrows mark features of BACs that have failed to recombine and Red arrows show features that indicate fragments that should be absent in the case of (LoxP) × (Lox5171) mistargeted recombination. Fragment list on left displays alterations to the reactant BAC NheI banding pattern that would occur in each of the three possible outcomes.
