Fig. 1.

Generation of Nedd4L conditional knockout in lung epithelia that express SPC. (A) General strategy for generation of floxed Nedd4L allele in mice, targeting the N-terminal region of the HECT domain (exon 15), to inactivate catalytic activity. After confirmation of proper homologous recombination by Southern blotting, the heterozygous mouse with the exon 15 flanked by two loxP sites (black triangles) and the neomycin resistance cassette (Neo) flanked by two FRT sites (gray boxes) [Floxed allele (+Neo)] were crossed with the FLP deleter mouse to generate heterozygous Nedd4L^f/+ mouse without the neomycin resistance cassette [Floxed allele (−Neo)]. This mouse was crossed with the SPC^rtTA;tet^on-Cre mouse to conditionally inactivate the Nedd4L allele (deleted allele). (B) PCR analysis of genomic DNA to test for the presence of Nedd4L floxed (f) allele, SPC, or Cre. In this example, only mouse 13 was a knockout, expressing Nedd4L^f/f, SPC, and Cre. (C) RT-PCR of isolated alveolar-type II cells of P14 mice proving loss of the N-terminal region of the HECT domain, as also verified by sequencing. Removal of exon15 causes a frameshift that deletes the whole HECT domain. (D) Loss of exon 15 in lungs from E18.5 knockout embryos analyzed by PCR on genomic DNA and verified by sequencing. (E) Survival rate of Nedd4L^f/f;SPC-rtTA;tet^o-Cre KO mice and controls (all other phenotypes), revealing death of most of the KO mice at 2–3 wk of age (n = 37 for Nedd4L^f/f;SPC-rtTA;tet^o-Cre and 109 for the controls). Some of the KO mice died immediately after birth from an unknown cause (not included in the figure).