Targeted disruption of <i>Med28</i>.

<p><b>(A)</b> Schematic representation of the targeted allele and null allele. Targeted <i>Med28</i> allele in mouse ES cells with neo cassette in the reverse orientation (top, Med28(fl-frt), targeted <i>Med28</i> allele without neo cassette after transient transfection with Flp (middle, Med28(fl)) and Med28 null allele with Exon 1 and 2 deletions after breeding with Protamine-Cre mouse (bottom, Med28(-)). Shaded boxes with numbers represent the exons. Open triangle: loxP site; closed triangle: Frt site; B: BspH1 restriction site; S: SpHI restriction site. Numbered arrows represent PCR primers used for genotyping. Short solid bar represents 5’ probe outside the homologous region used for Southern blot analysis. <b>(B)</b> Representative Southern blot analysis used to confirm correct targeting. DNA was digested with either BspHI or SpH1 restrict enzymes and the 5’Probe (shown in <b>A</b>) outside the homologous region was used to detect wildtype (+), floxed (fl) and null (-) alleles at expected sizes for WT (+) allele, 14.6 kb (BspHI) or 15.2 kb (SpHI); floxed allele without neo (fl), 15 kb (BspHI) or 12 kb (SpHI); and null (-) allele, 12.1 kb (BspHI) or 12.7 kb (SpHI). <b>(C)</b> Representative PCR analysis using primers 1 and 2 (shown in A) confirm the presence of 5’loxP site (top panel, 324 bp) and WT allele (top panel, 290 bp). PCR analysis using primers 1 and 3 (shown in <b>A</b>) confirm presence of only the null (-) allele (bottom panel, 310 bp). <b>(D)</b> Histological analysis of 6.5-dpc (E6.5) presumed to be Med28 mutant embryos shows disorganized extraembryonic tissue with no discernible epiblast. Sections from whole decidual swellings were stained with hematoxylin and eosin. Arrow, epiblast; arrowhead, trophoblast giant cell.