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. 2014 Mar 15;25(6):841–851. doi: 10.1091/mbc.E13-09-0543

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© 2014 Liu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Generation of Borg5-null mice. (A) The expression of Borg5 in mouse embryonic heart. Confocal images of wild-type E16.5 heart sections stained with Borg5 rabbit polyclonal antibodies together with antibodies to PECAM-1 or Nkx2.5. Borg5 was found exclusively in ECs labeled with PECAM-1 but not in cardiomyocytes labeled with Nkx2.5. Scale bars: 100 μm. (B) Gene-targeting strategy. The entire coding sequence of Borg5 (light gray arrows) together with part of the intron sequence was replaced by a neomycin-resistance gene (Neo). The noncoding regions of the exons are illustrated as dark gray bars. (C) Mouse tail PCR of the wild-type and knockout alleles produced DNA fragments of 454 base pairs and 578 base pairs, respectively. (D) Immunofluorescence staining of frozen heart sections from wild-type (+/+) and Borg5-null (−/−) embryos at E15.5 using the rabbit Borg5 antibody. No specific Borg5 staining was found in the Borg5-null (−/−) mouse heart. Scale bar: 50 μm. (E) Western blotting analysis of primary MCEC lysates using a Borg5 antibody generated in chicken. The lack of Borg5 protein in Borg5-null (−/−) MCECs confirms the correct targeting. Actin was used as a loading control. Similar results were obtained with the rabbit antibody for Borg5. (F) Wild-type and Borg5-null littermates at E16.5. No gross developmental defects were observed in the mutant. Scale bar: 1 cm. (G) Offspring of each genotype recovered from Borg5^+/− mating pairs. A significant reduction of Borg5^−/− mice was observed at P0 and at weaning. P0, total pups recovered at birth. P0 live, live pups recovered at birth. Statistical analysis by chi-square test, *, p < 0.05; **, p < 0.01.