Figure 2.

In vivo peptide nucleic acid (PNA)-Antp mediated gene targeting. (a) Mice were intraperitoneally (i.p.) injected with Rhod-167-PNA or Rhod-167-PNA-Antp. Tissues were harvested and visualized by fluorescence microscopy. (b) AV mice containing the chromosomally integrated supFG1 reporter gene and the control cII gene. The 167-PNA-Antp conjugate was designed to bind to the homopurine strand at positions 167–176 of the supFG1 gene. In this structure, one strand forms Watson–Crick hydrogen bonds with the purine-rich strand of the target duplex, while the other PNA strand forms Hoogsteen base pairs to the purine DNA strand in the PNA-DNA duplex. This strand invasion complex results in the displacement of the other DNA strand to form a D-loop, generating a PNA:DNA:PNA triple helix. (c) AV mice were treated with 167-PNA, 167-PNA-Antp, or phosphate-buffered saline (PBS). Tissue samples were harvested and analyzed by vector rescue from genomic DNA for analysis of the supFG1 reporter gene. Mutation frequencies were calculated and the combined totals from all tissues tested are displayed. Targeting specificity was evaluated by analysis of mutation frequencies in the nontargeted control gene, cII. Sequence analysis of the supFG1 gene mutations induced by treatment with 167-PNA-Antp further confirming site-specificity. The target site is underlined and (+) and (−) represent single base pair insertions and deletions. (d) Targeted mutagenesis of the supFG1 gene in selected somatic tissues of AV mice. Antp, antennapedia.