Generation of aspa^lacZ mice by homologous recombination.

A, Genomic structure of the murine aspa locus, which spans 6 exons (black boxes). Homologous recombination of the targeting vector inserts the βgeo cassette (blue box), encoding β-Galactosidase (lacZ), into intron 1 of the intact aspa gene. This cassette is flanked by frt-sites (green circles). Additional loxP sites flank exon 2 (red triangles). The vector includes a DTA cassette for negative selection. In the targeted allele, exon 1 is spliced to the splice acceptor site preceding βgeo, and transcription is terminated at the introduced pA site. The conditional aspa^flox allele is produced by breeding aspa^lacZ mutants to FLP-deleter mice [40] for recombination of the βgeo cassette in vivo. Note that frt and loxP sites are not drawn to scale. βgeo, β-galactosidase-neomycin resistance cassette; SA, splice acceptor; pA, polyadenylation site, DTA, Diphteria toxin gene. B, For Southern blot analysis genomic DNA was digested with SmaI/ApaI. The neo probe (white box in A) detected the expected 7.5 kb band in heterozygotes and homozygotes. C, Genomic PCR of littermates with primers 1 & 3 produces the expected amplicons in aspa^+/+ (336 bp), aspa^lacZ/+ (387 bp and 336 bp) and aspa^lacZ/lacZ (387 bp) animals. The upstream loxP site was detected by PCR with primers 1 & 2 yielding a 307 bp band. D, Q-RT-PCR using a TaqMan probe for quantification of aspa mRNA levels in brains of aspa^+/+, aspa^lacZ/+, and aspa^lacZ/lacZ mice (n = 3) confirms the attenuation of transcription downstream of exon 2 in the targeted allele. E, Representative Western blot of whole brain lysates of aspa^+/+, aspa^lacZ/+ and aspa^lacZ/lacZ mice (aged 4 months, n = 3). The 37 kD protein ASPA was detected in aspa^+/+ and aspa^lacZ/+ mice but not in aspa^lacZ/lacZ mutants. β-Galactosidase is expressed in aspa^lacZ/+ and aspa^lacZ/lacZ brain but not controls. α-Tubulin was used as loading control. F. Representative picture of a male aspa^lacZ/lacZ mutant (asterisk) and an aspa^+/+ littermate at P70.