Targeted disruption of the mouse Ikbkap gene.

<p>(<b>A</b>) Schematic representation of wild-type allele (WT), Ikbkap targeting vector (vector), and Ikbkap targeted allele (3loxP). Exons are represented by black rectangles, and black ovals represent the loxP sites. The position of the 5′ external probe along with the sizes of the diagnostic fragments are indicated. The positions of the primers used for genotyping are indicated by numbered arrows (1, 2, 3, 4), also the position of the pgk oligo is marked by an arrow above the neo cassette. A loxP flanked neostop cassette (neo HIS3-stop) is also indicated. Restriction sites shown on the schematic are: Bam HI (B), HindIII (H), BstXI (BX) and BstEII (BE). (<b>B</b>) Southern blot of WT (lanes 1,4, 5, and 7) and targeted (lanes 2,3, and 6) ES clones, DNA digested with BamHI and probed with the 5′ flank probe. (<b>C</b>) Genotyping of progeny derived from Ikbkap^3loxP/+ intercross. Genomic DNA from WT (lane 1), heterozygous (lane 2) and homozygous (lane 3) progeny was amplified by PCR using the primers Ikap1for and Ikap2rev 2. (<b>D</b>) RT-PCR on reverse transcribed RNA from WT (lanes 1 and 2) and Ikbkap^3loxP/3loxP (lanes 3 and 4) embryos using the primers Ikap18for and Ikap23rev is depicted (top panel). β-actin amplification (bottom panel) was performed in parallel to verify that equal amounts of starting material were used in each reaction.