Figure 7: Conditional deletion of the ANXA8 allele.

(a) (1) ANXA8 wt locus; (2) targeting vector (without negative selection marker and plasmid backbone); (3) genomic locus after homologous recombination (intron 1 containing the neomycin cassette, LoxP-flanked exon 2); (4) after removal of exon 2 by crossing with a pgk-Cre mouse. Numbered filled boxes represent exons, and lines represent intronic and intergenic regions. The empty box corresponds to the neomycin resistance cassette (neo) flanked by the FRT sites (not shown). Arrows indicate the LoxP sequences, and EcoRI (R) and HindIII (H) restriction sites. The black box represents the Southern probe (HR). (b) Southern blot analysis of ES cell colonies. Clones 1H6 and 4F5 contain the targeted (fl) ANXA8 gene. (c) Southern blot analysis of tail biopsy DNA isolated from wt (W1, W2, W3 and W4), ANXA8 targeted (fl; 145, 146, 148 and 149), and ANXA8 exon 2-deleted (Δ; 5, 8, 22 and 33) animals. Additional small bands are because of incomplete digestion at the left HindIII site and hence generation of fragments extended by 261 bp. (d) Genotyping PCR on tail genomic DNA. M, molecular weight standard; NC, negative control PCR without genomic DNA; +/+, wt animals; fl/fl, homozygous animals carrying the ANXA8 floxed allele; +/fl;pgk-Cre, offspring after crossing homozygous targeted animals with pgk-Cre mice; fl/fl;pgk-Cre, homozygous KO mice. (e) Detection of anxA8 protein expression in wt (+/+) and KO (fl/fl;pgk-Cre) mice by immunoblotting. α-Tubulin served as loading control. Note that expression of anxA2 is unchanged.