Figure 1.

Generation of AU040320-targeted mice. (A) Schematic representation of AU040320-targeting strategy. A “knock-out first” (tm1a or KO1) allele was generated by EUCOMM in ES cells after integration of a targeting vector containing exon 3 and ~4 kb flanking fragments each side, with 2 small regions replaced by a trapping cassette (containing 2 loxP sites (P1, P2) and 2 FRT sites (F1, F2)) and a loxP-cassette (containing a loxP site (P3)), respectively. Live mice carrying this allele were obtained and used to generate other alleles (full details in Materials and Methods section). A “del” (tm1b) allele was obtained by deletion of the P1-to-P3 region after Cre recombination. After Flp recombination on the tm1a allele, a “floxed” (tm1c or Flx) allele was generated by deletion of the F1–F2 region. The Flx allele has conditional knockout potential, with the removal of the critical region containing exon 3 between sites P2 and P3 to obtain a “Null” (tm1d or Null) allele after Cre recombination. A long-range PCR fragment (KO1-5′) used for target confirmation in tm1a allele, and the genotyping PCRs used for identification of the different alleles are represented. Details about elements in the targeting cassette and the different PCRs are shown in Fig. S2 and Table S2. (B) Long-range PCR KO1-5′ from KO1 homozygous mouse confirming specific insertion of targeting construct in the AU040320 locus. DNA ladder and size of 2 bands are shown on the left. (C) Results of genotyping PCRs from mice homozygous for wild-type (ww), KO1 (aa), del (bb), Flx (cc) or Null (dd) alleles; heterozygous Flx (cw) is included to show the expected double band with PCR KLWF. Size of fragments (bp) appears on the right. (D) Western blotting analysis from mouse brain lysates with specific antiserum KL-FCt-G1 (#78). Comparison of samples from wild-type and all 4 homozygous AU040320-targeted adult (16–24 week-old) male mice (top panel) shows that the AU040320 protein (arrow), with an apparent ~150 kDa size, is clearly detected in wild-type (w/w) and Flx (c/c), heavily reduced in KO1 (a/a) and totally absent in del (b/b) and Null (d/d) samples. The ~65 kDa band detected at the bottom of the picture is the strongest of several unspecific bands detected by this antibody (see Fig. S1A). Same results were obtained with samples from female mice (not shown). The AU040320 protein is detected in brain samples from wild-type mice at different developmental stages (bottom panel), including embryonic days 15 (E15) and 18 (E18), and a wide range of postnatal days (P1-to-P159); adult Null (left) and del (right) samples are shown for comparison purposes. 30 μg total protein loaded per lane.