Fig 1. Gene targeting and analysis of targeted integrants.
(a) Map of Muc5b recombined and Muc5b KI loci, and after Flpe-mediated excision of the positive selectable neomycin (Neo) marker. The FRT (F, vertical purple lines) and loxP (L, vertical red lines) sites are indicated. The loxP sites were not used in this study. The NsiI and NheI restriction sites used for Southern blot experiments are shown. The NheI restriction within intron 47 does not exist in the wild-type (WT) allele. Exons are indicated by rectangles and few of them are numbered. The unique 3’ untranslated region (UTR) is shown in black and belongs to the 49th and last exon. (b) Southern blot analysis of homologous recombination in nine embryonic stem (ES) cell clones. Genomic DNA of the ES cell clones was compared with WT DNA (129SvPas ES cells). Correct targeting by homologous recombination is indicated by a 13.2-kb band obtained with a Neo probe on NsiI digested DNA whereas no band is visible for the WT as expected. (c) A 11.6-kb and 14.5-kb band obtained with the external 3’ probe on NheI-digested genomic DNA for the recombined locus and the WT allele, respectively.