Figure 3. Targeting of the miR-126 gene.

(A) Strategy to generate miR-126 mutant mice by homologous recombination. The 96bp Egfl7 intron 7 sequence, which contains miR-126, was replaced with a neomycin resistance cassette (Neo) flanked by loxP sites. Neo was removed in the mouse germ line by crossing heterozygous mice to CAG-Cre transgenic mice. DTA, diphtheria toxin A.

(B) Southern blot analysis of genomic DNA from ES cells. DNA was digested with Sca I. Using either the 5’ probe or 3’ probe, the sizes of the wild-type and mutant (miR-126^neo allele) are 11.4kb and 13.4kb, respectively. Genotypes are shown on the top.

(C) Analysis of Egfl7 transcripts in heart (left panel) or lung (right panel) of miR-126^neo/neo or miR126^−/− mice, as detected by RT-PCR. The Egfl7 gene structure and the exon numbers are shown on the bottom. Primers used for RT-PCR were named based on the exon number in the forward (F) and reverse (R) direction. Genotypes are shown on the top. GAPDH was used as a control. Note that Egfl7 expression is disrupted in the miR-126^neo/neo mutants, as shown by RT-PCR with primers 7F and 8R, 6F and 9R, 8F and 10R; and normalized upon deletion of the neo cassette, as shown by RT-PCR with the primers indicated.

(D) Detection of EGFL7 and GAPDH protein by Western blot of heart extracts from WT and miR-126 KO mice.

(E) Detection of miR-126 transcripts by Northern analysis of hearts and lungs. 5S rRNA serves as a loading control.

(F) Genotypes of offspring from miR-126^+/− intercrosses. The actual and expected number of mice for each genotype at the indicated stages is shown.

(G) Genotypes of embryos from miR-126^+/− intercrosses. The number of miR-126^−/− offspring analyzed at each age is shown. Severe vascular defects were defined as edema, hemorrhage, severe growth retardation and lethality. Less than 1% of wild-type or miR-126^+/− embryos or neonates showed vascular abnormalities.