Postnatal Mortality and Stunted Growth in <i>K14-cre; Apc^CKO/CKO</i> Mutant Mice

<div><p>Animals whose genotype is either heterozygous or homozygous for the wild-type <i>Apc</i> allele are referred to as normal (N); those whose genotype are <i>K14-cre; Apc^CKO/CKO</i> and show the presence of <i>K14-cre</i>–recombined mutant <i>Apc</i> allele are called mutant (M).</p><p>(A) Two P3 mutant mice, M1 and M2, and their normal littermates, showing size variation among mutants.</p><p>(B) P8 mutant mouse (right) and a normal littermate. Note sparseness of hair coat and abnormal ears.</p><p>(C–D) Vibrissae of whisker pads are short and oddly angled in a P12 mutant mouse (C), relative to control (D). Note the lack of incisors in the mutant.</p><p>(E) A P17 mutant mouse (right) with its littermate. Its bare forehead, dorsal median line, and abnormal ears are evident.</p><p>(F) Growth curve of mutants and normal littermates. Mutants exhibit stunted growth, which became more prominent as they aged, and weigh significantly less than littermates from P8 (<i>p</i> < 0.05).</p><p>(G) Comparison of mutant and normal thymus from P3 mice. The mutant thymus (left) is dramatically smaller for its age compared to the normal littermate (right). The scale bar equals 1 mm.</p><p>(H) Skeletal preparations of normal (left) and mutant (right), showing differences in development of both incisor (I) and molar (M) teeth.</p></div

Expression of <i>Shh</i> and β-catenin Transcripts in Normal <i>(Apc^CKO/CKO)</i> and Mutant <i>(K14-cre; Apc^CKO/CKO)</i> Embryonic Skin

<p>(A–D) Section ISH with <i>Shh</i> probe in E14.5 normal (A), E14.5 mutant (B), E16.5 normal (C), and E16.5 mutant (D) skin. Broken lines indicate the interface between epithelium and mesenchyme. Scale bars: 50 μm. Whole mount in situ detection of β-catenin in E15.5 normal (E, G), mutant (F, H) embryos. Aberrant initiation of multiple hair placodes is evident at E14.5. Loss of <i>K14</i>-driven <i>Apc</i> loss caused aberrant pattern formation (F′) and formed ectopic hair placodes in normally hairless foot pads (H, arrows) which are absent in normal (G).</p

Tissue-Specific Detection and Expression of Deleted <i>Apc</i> Alleles

<div><p>(A) Tissue-specific genotyping PCR. Only genomic DNA samples from the skin (S) and thymus (T), but not liver (L) of mice positive for <i>K14-cre</i> show the presence of deleted <i>Apc^Δ580</i> allele.</p><p><b>(</b>B<b>)</b> Genotype- and tissue-specific expression of the truncated <i>Apc</i> transcripts. A representative gel of RT-PCR using primers F546 and R721, showing that only RNA from the skin and thymus but not liver of mice positive for <i>K14-cre</i> have transcripts from both wild-type (528 bp) and deleted (313 bp) <i>Apc</i> alleles.</p></div

Histological and Immunochemical Examination of Thymus

<p>(A–D) P3 normal thymus. (E–G) Mild P3 mutant thymus. (H–K) Severe P3 mutant thymus. (L–O) P13 mutant thymus. Stained with H&E for histology (A, E, H, L), BrdU (B, I, M), β-catenin (C, F, J, N), and K14 (D, G, K, O). (B) Actively dividing thymocytes are visible at the superficial edge of cortex of normal P3 thymus. Note the progression of histological abnormalities in the mutant thymus from mild P3, severe P3 to P13 (A, E, H, L). Scale bars, 20 μm.</p

Generation of the Conditional <i>Apc</i> Allele

<div><p>(A) Schematic diagram of exons 14 and 15 of the mouse <i>Apc</i> gene, the targeting vector, and the resulting conditional allele with 2 LoxP sites sandwiching the exon 14. The PGK-neomycin cassette was inserted within intron 14 by recombineering technique. This cassette is sandwiched by 2 FRT sites that could be removed by crossing to FLPe-expressing mice. Positions of PCR primers used for genotyping PCR (F2, R2, R4) and RT-PCR (F546 and R721) are indicated. Positions of probe used for Southern blot analysis with NdeI sites are also shown. Upon Cre-mediated recombination, exon 14 is removed and leads to truncated Apc protein, of which the first 580 aa correspond to the normal.</p><p>(B) Southern blot analysis of NdeI-digested genomic tail DNA isolated from F1 mice of various <i>Apc</i> mouse lines <i>(Apc^CKON, Apc^Δ580),</i> hybridized to a 600-bp probe. Tail genomic DNA from <i>Apc^CKON</i> F1 mice derived from a modified ES clone showed a 12-kb band for the <i>Apc^CKON</i> allele and a 10-kb band for the wild-type allele, whereas genomic DNA from the <i>Apc^Δ580</i> mouse was heterozygous for the <i>Apc^Δ580</i> allele (9.2-kb band).</p><p>(C) Kaplan-Meier survival plot of <i>Apc^CKO/+</i> mice (thin solid line, <i>n</i> = 39), <i>Apc^CKO/CKO</i> mice (thin dotted line, <i>n</i> = 57), <i>Apc^Δ580/+</i> mice (solid line, <i>n</i> = 51), and wild-type littermates (broken line, <i>n</i> = 21). Heterozygosity of the <i>Apc^Δ580</i> allele led to a significantly shortened survival (<i>p</i> < 0.0001), whereas those of heterozygous and homozygous <i>Apc^CKO</i> mice had no significant difference to that of wild-type littermates.</p></div