Fig. 2.
Conditional deletion of Hox11 function recapitulates the germline null mutation. (A) Schematic illustrating the Hoxd11 locus. Yellow arrowheads illustrate the inserted loxP sites. Two guide RNAs with the indicated sequences (underlined) along with their corresponding PAM (highlighted blue) were used to flank exon 2 of Hoxd11 in order to insert loxP sites. Homology sequences used in the donor sequences are highlighted with thick dark blue line (5′ loxP) and thick light blue line (3′ loxP). Red arrows marked with P1, P2, and P3 mark the location of the PCR primers used to confirm recombination. Corresponding PCR product sizes are indicated as well. The PCR elongation time was adjusted so that a 300-bp PCR product would appear only if recombination had occurred between the loxP sites. Pregnant dams were fed on tamoxifen chow for 1 wk to induce recombination, and the resulting embryos were collected at E17.5. (B) PCR analysis using the PCR primers produces a robust 600-bp control band only present in the controls and a 300-bp recombined band only present in the conditional mutants. cKO denotes Hox11 conditional mutants. Skeletal preparations of limbs from (C) WT, (D) littermate control for Hox11 conditional mutant, (E) Hox11ROSACreERT2 conditional mutant, and (F) Hox11 germline-null mutant. The red box highlights the zeugopod skeleton.