Figure 1.

Generation and validation of miR-200b^−/− mice (miR200b^{tm1.1(NCOM)MFGC}). (a) Targeted miR-200b knockout allele. The complete miR-200b non-coding gene (WT allele) was replaced by a NorCOMM cassette via targeted homologous recombination in C57Bl/6N mouse C2 ES cells. The NorCOMM targeting cassette consists of three functional components: a lacZ expression reporter (blue boxes); a loxP flanked (red triangles) hβact promoter driven ∆TK1-T2A-neomycin selectable marker (orange boxes), which can be subsequently excised by cre-recombinase; and a docking cassette AttP-Puro-pA that can be utilized to exchange the entire NorCOMM cassette to any other allele (purple boxes) and once placed, the remaining flanked sequences can be removed by flpO-recombinase between F3 and FRT sites (green triangles) in vivo. The length of 5′ and 3′ homology arms are 2891 bp and 6595 bp in the targeting vector, respectively. The targeted miR-200b gene was highlighted in the resulting knockout allele (KO Allele). (b) Targeted miR-200b knockout mice. Removal of the hβact promoter driven ∆TK1-T2A-neomycin cassette was performed by mating male miR200b^{tm1(NCOM)MFGC} mice harboring the miR-200b KO allele with female CMV-Cre transgenic mice. Precise cre-excision of the neomycin cassette was determined by PCR and sequence validation, as shown with primers specific to LacZ and Puro cassette by purple arrows in Panel C. The resultant miR-200b cre-excised allele is shown as miR200b^{tm1(NCOM)MFGC}. (c) LacZ staining of miR-200b^−/− mice demonstrated the expression in both epithelial and mesenchymal cells during lung development. (d,e) Lung explants culture of E11.5 lung explants did not show significant differences in proximal branching morphogenesis between miR-200b^+/+, miR-200b^+/− and miR-200b^−/− lungs. (f) RT-qPCR for all miR-200 family members on fetal lung explants using LNA primers. miR-200b absence was confirmed. miR-200a and miR-429 were significantly downregulated, but no changes were observed in abundance of miR-200c and miR-141 **P < 0.01, Student’s t-test, Data represent mean ± SEM of at least four independent experiments.