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. 2011 May 20;6(5):e20336. doi: 10.1371/journal.pone.0020336

Figure 1. Generation of aspalacZ mice by homologous recombination.

Figure 1

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 aspaflox allele is produced by breeding aspalacZ 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), aspalacZ/+ (387 bp and 336 bp) and aspalacZ/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+/+, aspalacZ/+, and aspalacZ/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 +/+, aspalacZ/+ and aspalacZ/lacZ mice (aged 4 months, n = 3). The 37 kD protein ASPA was detected in aspa +/+ and aspalacZ/+ mice but not in aspalacZ/lacZ mutants. β-Galactosidase is expressed in aspalacZ/+ and aspalacZ/lacZ brain but not controls. α-Tubulin was used as loading control. F. Representative picture of a male aspalacZ/lacZ mutant (asterisk) and an aspa+/+ littermate at P70.