Figure 1.

Breeding design for production of Hoxb1^A1 and control founders. (A) To produce animals bearing Hoxb1^A1swaps that also possess the functional behaviors needed for OPAs, Hoxb1^A1(g)/A1(g) 129 × C57BL/6 mice were bred to outbred, wild-derived mice. The resulting Hoxb1^A1(g)/+ heterozygotes were crossed to establish the next (F₂) generation. Progeny were genetically screened and only Hoxb1^A1(g)/A1(g) individuals were selected as OPA founders. Three OPA populations were established with these F₂ animals and three more were established with F₃ animals produced from F₂ Hoxb1^A1(g)/A1(g) homozygous breeding pairs. (B) To control for potential confounding effects due to differential genetics surrounding the swap control animals were bred by crossing Hoxb1^+(g)/+(g) 129 × C57BL/6 mice with the same wild stock used in the Hoxb1^A1 treatment lineage. The Hoxb1^+(g)/+ were then crossed to produce the F₂ generation. Only Hoxb1^+(g)/+(g) mice were selected as OPA founders. Three OPA populations were established with these F₂ animals and three more were established with F₃ animals produced from F₂ Hoxb1^+(g)/+(g) homozygous breeders. (C) Illustrations of wild type (Hoxb1⁺), wild type with the IRES–τ-GFP tag (Hoxb1^+(g)), and Hoxb1^A1 swap with the IRES–τ-GFP tag (Hoxb1^A1(g)) are provided. Large rectangles represent exons 1 and 2 of Hoxb1 (black) and Hoxa1 (white). The Hoxb1 promoter is conserved across all genotypes and solid squares represent the Hoxb1 autoregulatory enhancer. Loops separating the τ-GFP tag from the second exon depict the IRES. Arrows approximate primer binding sites. Illustrations are not to scale. (D) Image of polyacrylamide gel discrimination between Hoxb1^A1(g) and Hoxb1⁺ (left) and between Hoxb1^+(g) and Hoxb1⁺(right) alleles.