FIG. 3.

Disruption of PDX2. (A) Schematic describing the disruption plasmid. A clone was constructed in which the PDX2 inverse PCR product, which consists of the 5′ and 3′ ends of PDX2 separated by both 5′ and 3′ flanking sequences, was ligated to pBARKS1, a bialophos resistance-encoding vector. Standard gene disruption constructs that rely on double-crossover events for successful gene replacement generally contain an antibiotic resistance gene within the gene being disrupted. This plasmid corresponds to a standard gene disruption construct except that the entire vector containing the antibiotic resistance marker has been used to interrupt PDX2. (B) Southern hybridization analysis of a pdx2-disrupted strain. EcoRI-digested DNA from the wild-type (WT) and a pdx2-disrupted (DISR.) strains was probed with a PDX2-specific probe spanning the entire ORF. In the wild-type strain, the entire ORF is contained on a single EcoRI fragment, which is split into three parts in the gene disruption strain.