Figure 1.

Strategy to generate a AQP0^ΔC/ΔC by introducing a stop codon after amino acid 246. (A) WT: Schematic structure of WT mouse AQP0 gene showing exons 1-4 (as rectangular vertical or horizontal boxes) and the connecting introns. Vector: Exons 3 and 4 with introns (highlighted in blue and red) as well as a Neo selection gene were amplified by PCR and cloned into the vector (details in [B]). Black dotted lines on either side denote vector sequences. Asterisk indicates an in-frame translation stop codon predicted to truncate AQP0 after the amino acid Asparagine-246. KI-Neo: The recombinant vector (with Exons 3 and 4, introns highlighted in blue and red and the Neo selection gene) was transfected into mouse embryonic stem cells and positive clones were selected using the Neo selection marker. KI: KI with stop codon after amino acid 246 but without the Neo-Selection marker. Light green vertical rectangle indicates one set of the LoxP-FRT sites that remain after Neo deletion (74 bp). Positive embryonic stem cells selected were injected into mouse blastocysts to develop AQP0 KI mouse model (AQP0^ΔC/ΔC). (B) Schematic of the introduction of the stop codon through a point mutation incorporated into a primer pair (to delete the 17 amino acids after the 246^th), and the KI targeting vector design. The C-terminal end deletion was engineered by overlap extension PCR using the primers as indicated. Red rectangle represents the deletion region. SA, short homology arm; LA, long homology arm. N1, N2, PT2, PT3, PT4, P6, and T73 are forward or reverse primers used, as indicated.