Fig. 1. Generation and validation of Egfl7 and miR-126 deletion alleles. (A) Egfl7 and miR-126 delta () alleles were generated by flanking exons 5-7 of Egfl7 or a 289 bp segment of intron 7 containing miR-126 with loxP sites, respectively, followed by in vivo deletion using Cre recombinase. Green arrowheads, remnant loxP sites after Cre deletion. Blue arrows, PCR primers used in E. Red line, Egfl7 epitope used for polyclonal antibody generation. (B) In situ hybridization for processed miR-126 (dark purple staining) demonstrates vascular expression in the trunk region of wild-type (wt) E14.5 mouse embryos (top panels) that is absent in miR-126^/ embryos (bottom panels). Arrows in higher magnification images (taken from the boxed regions) highlight vascular miR-126 expression in the neural tube and carotid artery in wild-type embryos, and arrowheads the absence thereof in miR-126^/ embryos. CA, carotid artery; JV, jugular vein; NT, neural tube; OE, esophagus; TR, trachea; VA, vertebral artery. (C) Quantitative PCR (n=6) confirmed the absence of Egfl7 mRNA in Egfl7^/ embryos and the absence of mature (processed) miR-126 in miR-126^/ embryos. A looped RT primer specifically detecting the mature miR-126 processed end was utilized. Notably, Egfl7^/ embryos exhibited normal miR-126 processing and miR-126^/ embryos exhibited normal levels of Egfl7 mRNA, indicating that microdeletion did not disrupt physiological expression of the adjacent gene/miRNA in either case. ^*P<0.001 versus wild type. (D) Immunofluorescence staining of uterus from a pregnant mouse with affinity-purified rabbit anti-Egfl7 antibody demonstrating loss of Egfl7 protein in adult Egfl7^/, but not adult miR-126^/, mice. (E) RT-PCR of full-length Egfl7 coding sequence from miR-126^/ embryos indicates that microdeletion of miR-126 does not induce occult splicing of Egfl7 mRNA. A doublet is present in Egfl7^/ embryos representing out-of-frame splicing from exon 4 to exon 8 or 9.