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Fig. 1. Vax2 mRNA expression in the mouse and inactivation of the mouse Vax2 gene. (A) Whole-mount in situ hybridization for Vax2 mRNA in an E9 mouse embryo. (B) High-power view of an E11.5 mouse eye, illustrating the pronounced dorsal-ventral (DV) Vax2 gradient. D, V, N, and T indicate dorsal, ventral, nasal and temporal poles of the retina. (C,D) Front (C) and back (D) views of a whole-mount in situ hybridization for Vax2 mRNA (purple reaction product) in a dissected mouse retina at E14. Arrowhead denotes a ventral cut made before the eye was dissected from the embryo. (E) In situ hybridization of a coronal section through a wild-type P0 mouse eye, demonstrating high expression of Vax2 mRNA in the retinal ganglion cells (rgc) of the ventral (V) but not the dorsal (D) retina. (F) In situ hybridization of a transverse section through a wild-type P0 mouse eye, demonstrating slightly higher expression of Vax2 mRNA in the retinal ganglion cells (rgc) of the nasal (N) than the temporal (T) retina. (G) Targeting construct for inactivation of the mouse Vax2 gene, and structure of the inactivated allele after homologous recombination in mouse embryonic stem cells. A BamHI/EcoRV genomic fragment containing exon 2 of the Vax2 gene was replaced by a PGK-neo cassette (see Materials and Methods). Exon 2 encodes amino acid residues 83-145; these residues include the first two {alpha} helices of the Vax2 homeodomain, which are essential to the function of all known homeodomain transcription factors. The BamHI/EcoRV deletion also introduces a shift in the Vax2 reading frame. (H) Southern blot of XbaI and SalI-digested genomic DNA from wild-type (+/+) and heterozygous (+/–) ES cell clones probed with the external 3' probe indicated in G and described in Materials and Methods. (I) RT-PCR analysis of mRNA isolated from wild-type (+/+) and Vax2 mutant (–/–) retinae at P0. The primers for the PCR reaction are located in exons 1 and 3, and correspond to nucleotides 127-148 and 586-606 of the mouse Vax2 cDNA sequence (Genbank Accession Number, Y17792). The size of the –/– fragment is consistent with the clean excision of exon 2, which was confirmed by DNA sequence analysis of the 468 and 261bp bands, and by the loss of Vax2 immunoreactivity in the homozygous mutant retina (data not shown). Scale bar: 0.1mm in E,F.