Targeting of the α-casein gene.
Panel A: Schematic representation of the murine casein locus. Casein genes are represented as solid arrows. Other predicted genes are shown as open arrows. Panel B: Schematic representation of the unmodified α-casein gene and the targeted α-casein gene. Exons of the α-casein gene are indicated as solid boxes, the hytk selection marker gene is indicated as striped box. The PGK promoter element directing expression of the selection marker gene is indicated as arrowhead. The relative positions of the EcoRI restriction sites (EI), the Southern blot probe (probe), sizes of hybridising DNA fragments and the primer binding sites used for genotyping (horizontal arrows) are indicated. Panel C: PCR analysis of genomic DNA isolated from the three representative ES cell clones using the primer combination acas6, acas7 and pBKpA2 (analysing the 5′ end of the homologous recombination event). A 1566 bp band is detected in all samples and represents the unmodified α-casein allele [U: unmodified]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 848 bp PCR product [T: targeted]. Marker: phage λ digested with HindIII and EcoRI (λ×H/E). Panel D: Southern blot analysis of EcoRI digested DNA derived from tail clips of a wild-type (acas [+/+]), and α-casein mutant mice (heterozygous: acas [+/−], and homozygous α-casein [−/−]). The probe indicated in panel B detects a 7.5 kb DNA fragment representative of the unmodified α-casein allele [U] and a 4.3 kb band representative of the targeted α-casein allele [T]. Panel E: PCR analysis of genomic DNA isolated from two ES cell clones using the primer combination acas1, acas7 and PGK5 (analysing the 3′ end of the homologous recombination event). A 688 bp band is detected in both samples and represents the unmodified α-casein allele [U]. The second clone carries a targeted α-casein allele as indicated by the occurrence of a 450 bp PCR product [T]. Marker: NEB PCR marker.