Figure 1. Generation of new conditional allele and functional confirmation of the Pitx2 muscle specific knockout mouse.

(A) Schematic representations of the endogenous Pitx2 locus, targeting vector, Pitx2^FL allele, and the Cre-recombined allele. Exon 5, encoding the upstream end of the homeobox, is flanked by loxP sites (grey arrowheads), while the PGKNeo (PGKN) cassette is flanked by FRT sites (pink arrowheads). Tissue-specific recombination with a Cre driver results in the Pitx2^Δ allele (bottom). (B) Homologous recombination in ES cells. Genomic DNA from ES cell clones was digested with BamH1 and probed with the 5’ probe shown above to identify homologous recombination with a13.5 kb fragment (C) Demonstration of Cre recombination of genomic DNA from tissues. The floxed (Pitx2^FL) and deleted (Pitx2^Δ) form of the allele are detected as 1400 and 360bp amplicons, respectively, in PCR amplifications of genomic DNA isolated from brain (Br), cardiac outflow track (H), liver (L), white adipose tissue (A), or muscles (TA, Ga) of P30 conditional null mice (Pitx2^MCK). Excision was only detected tibialis anterior (TA) and gastrocnemius (Ga). (D) Western blot of total protein extracts from the organs of normal and conditional null mice at P30. Pitx2 protein levels were reduced in the abdominal body wall (Ab) and muscles (TA, Ga) of conditional mutants but were unaffected in intestine (I). Intestinal Pitx2 protein levels are reduced in complete (Pitx2^Z/Z) but not conditional (Pitx2^MCK) mutants. (E-H) Triple labelling immunohistochemistry of E16 hindlimb muscle (E, F) and P2 tibialis anterior (G, H) of control MCK^cre|Rosa^nT-nG (E, G) and mutant MCK^cre|Rosa^nT-nG |Pitx2^FL/Z (F, H) mice. Onset of Cre excision is indicated by the onset of green signal in red nuclei (yellow). Loss of Pitx2 protein signal (blue) was observed in these cells.