Figure 9

Targeting scheme to generate the Gsx2^EGFPknock-in allele. (A) Using homologous recombination in embryonic stem cells an IRES-enhanced green fluorescent protein (EGFP) cassette was inserted in the first exon of the Gsx2 between an Nco 1 and Not 1 site. This removed 125 bp of the coding region of the first exon but left the exon-intron structure intact. The polyA signal at the end of the IRES-EGFP cassette effectively terminated the message and, as shown in Figure 1, no Gsx2 protein is observed when the Gsx2^EGFPallele is bred to homozygosity. The Neomycin (Neo) cassette was removed by breeding the mice with β-actin-Flpase mice [38]. These Gsx2^EGFPminus Neo mice were exclusively used in this study. (B) Correctly targeted embryonic stem cells were identified using the primers indicated as half arrows in (A). (C) Embryos are genotyped using primers to detect the Gsx2^EGFPallele and using Gsx2 primers that include one sequence in the deleted region of the first exon. M, DNA marker; pd, primer dimmer.