Figure 1.

Targeting strategies and expression of the Pax3 alleles. (A) Schematic diagram of the targeted Pax3^IRESnLacZ allele. The 5′ part of the Pax3 gene contains exons 1-4 (the gene spans 100 kb and contains eight exons). We targeted an IRESnLacZ sequence followed by the SV40 polyadenylation sequence and a floxed puromycin-pA selection cassette, into exon 2 of Pax3 by homologous recombination in embryonic stem (ES) cells. A counterselection cassette encoding the A subunit of Diphtheria Toxin (DTA) was inserted at the 5′-end of the vector. The probes and restriction enzymes are indicated, as well as the size of the resulting wild-type and recombined restriction fragments. We chose to target the nLacZ reporter gene into exon 2 in order to inactivate the paired DNA-binding domain. The Pax3^{IRESnLacZ(puro)} allele was crossed with a PGK-Cre mouse (Lallemand et al. 1998) to remove the Puromycin selection cassette, leaving a single LoxP site 3′ of IRESnLacZpA in the final Pax3^IRESnLacZ allele. (B) Schematic diagram of the targeted conditional Pax3^{PAX3-FKHR-IRESnLacZ} allele. The 5′ part of the Pax3 locus is indicated as in A. In the targeting vector, the floxed puromycin-pA selection marker replaces the coding sequence in exon 1 of Pax3, followed by a dicistronic cassette containing the human PAX3-FKHR coding region followed by an IRESnLacZpA cassette, in order to be able to follow the expression of the allele. The probes and restriction enzymes are indicated, with the size of the resulting wild-type and recombined restriction fragments. After homologous recombination, PAX3-FKHR-IRESnLacZ expression from the Pax3^{Puro(PAX3-FKHR-IRESnLacZ)} allele is blocked by the floxed puromycin-pA cassette and is therefore conditional to removal by crossing with a Cre mouse (Lallemand et al. 1998). As Pax3^{PAX3-FKHR-IRESnLacZ/+} heterozygotes are not viable, the mice were maintained as Pax3^{Puro(PAX3-FKHR-IRESnLacZ)/+}. (C,D) Expression of nLacZ revealed by X-Gal staining (D) compared with expression of endogenous Pax3, visualized by in situ hybridization (C), at E10.5 shows that IRESnLacZ is correctly expressed in the Pax3^IRESnLacZ/+ embryos, in the dermomyotome and myotome of the somites (black arrow), limb buds (red arrows) and hypoglossal chord (green arrow), fronto-nasal masses and olfactory epithelium, and dorsal neural tube (white star). (E) expression of Pax3^{PAX3-FKHR-IRESnLacZ/+} at E10.5 shows that the Pax3^{PAX3-FKHR-IRESnLacZ/+} allele is also expressed in somites (black arrow), limb buds (red arrows) and hypoglossal chord (green arrow), fronto-nasal masses and olfactory epithelium, and dorsal neural tube (white star).