Generation of <i>Snai3</i> mutant alleles.

<p>(<b>A</b>) Construction of the <i>Snai3</i> conditional null allele. Schematic representation of the wildtype <i>Snai3</i> allele, the <i>Snai3^flox-neo</i> targeting vector, and the targeted <i>Snai3^flox-neo</i> allele. Exons are indicated by rectangles with coding sequences designated by gray shading, and noncoding sequences in black. LoxP sequences are marked by black triangles and FRT sites by gray triangles. An FRT-neo-FRT-loxP- cassette was inserted between exons 1 and 2, and the second loxP site was inserted 5′ to exon 1. A diphtheria toxin (DT) cassette was included for negative selection of randomly integrated clones. Hybridization probes used for Southern blot analyses are indicated. Abbreviations: B, BamHI; E, EcoRI; H, HindIII; N, NcoI; Nh, NheI; Sa, SalI; Sp, SpeI. (<b>B</b>) Construction of the <i>Snai3-EYFP^neo</i> knock-in allele. Schematic representation of the wildtype <i>Snai3</i> allele, the <i>Snai3-EYFP^neo</i> targeting vector, and the targeted <i>Snai3-EYFP^neo</i> allele. Exons are indicated by rectangles with coding sequences designated by gray shading. FRT sequences are marked by gray triangles. An EYFP-FRT-neo-FRT cassette was inserted at the ATG site in exon1, replacing the remainder of the exon1 coding sequence. A diphtheria toxin (DT) cassette was included for negative selection. Hybridization probes used for Southern blot analyses are indicated. Restriction enzyme abbreviations are as in A. (<b>C</b>) Absence of <i>Snai3</i> RNA expression in <i>Snai3^null/Snai3^null</i> and <i>Snai3-EYFP</i>/<i>Snai3-EYFP</i> embryos. Quantitative RT-PCR of relative <i>Snai3</i> transcript levels in RNA isolated from <i>Snai3^null/Snai3^null</i>, <i>Snai3-EYFP</i>/<i>Snai3-EYFP</i> and littermate control embryos revealed the absence of <i>Snai3</i> transcripts in the homozygotes, confirming that both alleles were <i>Snai3</i> null alleles.