TCGTCATC. The complete PCR product was cloned into an entry vector with BP clonase (Invitrogen), subsequently cloned into pDEST42 with LR clonase (Invitrogen) and eventually sequenced in its entirety. To produce recombinant LCN7 in BL21-DE3 cells, LCN7 expression was induced with IPTG and the resulting protein purified from bacterial cell lysates first on a Ni-agarose affinity column and second on an anti-FLAG M2 affinity column using standard procedures. Protein purity was monitored by coomassie staining of SDS-PAGE gels. Morpholinos and morpholino injections The LCN7 specific anti-sense morpholino (Genetools) (59TTAAACTCACTGGGAGGGTAGGGAG) was designed to interfere with splicing at the LCN7 exon1 – intron1 junction. The mismatched control LCN7 morpholino (59GTAAACGCACTGC- GAGGGTTGGGAA) was identical to the specific LCN7 morpho- lino except for the presence of 5 mismatched bases (underlined) that disrupt efficient binding to the LCN7 exon1 – intron1 splice junc- tion. Morpholinos were dissolved in water and diluted 1:1 into 0.1% phenol red/water injecting solution. Injections into 1 to 8 cell Fli1- GFP embryos ranged from 1 to 8 ng of LCN7 morpholino in a total volume of 0.3 to 0.6 nl per embryo. Approximately 28 hours after fertilization, embryos were sedated in tricaine and monitored for vascular phenotypes on a Nikon SMZ-1500 fluorescent dissecting microscope. Morpholinos directed against p53 were synthesized according to previously published work [22]. Plasmids and Retroviral Infections A retroviral LCN7 vector was constructed by PCR amplifying the full-length murine LCN7 cDNA from clone ID 3596560 (open biosystems). To accomplish this, oligos were designed such that an ATTB1 site was appended to the 59 end and the FLAG epitope tag and ATTB2 sequences were appended to the 39 end through two rounds of sequential PCR with the following oligos. First forward LCN7 oligo: AAAGCAGGCTTCACCATGTGGGGATGTTGG- CTG. Second forward LCN7 oligo: GGGGACAAGTTTGTA- CAAAAAAGCAGGCTTCACCATG. First reverse LCN7 oligo: CTACTTATCGTCGTCATCCTTGTAATCGTGGTGCCCCA- TGTCCTC. Second reverse LCN7 oligo: GGGGACCACTTTG- TACAAGAAAGCTGGGTCCTACTTATCGTCGTCATC. The PCR product was cloned by BP clonase into an entry vector and References 1. Maquart FX, Pasco S, Ramont L, Hornebeck W, Monboisse JC (2004) An introduction to matrikines: extracellular matrix-derived peptides which regulate cell activity. Implication in tumor invasion. Crit Rev Oncol Hematol 49: 199–202. 2. Liotta LA, Kohn EC (2001) The microenvironment of the tumour-host interface. Nature 411: 375–379. 3. Bornstein P, Sage EH (2002) Matricellular proteins: extracellular modulators of cell function. Curr Opin Cell Biol 14: 608–616. 4. Sottile J (2004) Regulation of angiogenesis by extracellular matrix. Biochim Biophys Acta 1654: 13–22. 5. Flower DR, North AC, Sansom CE (2000) The lipocalin protein family: structural and sequence overview. 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LCN7 Promotes Angiogenesis subsequently cloned with LR clonase into a custom made pMSCV retroviral vector featuring the ATTR1-CMr-ccdB-ATTR2 cassette blunt cloned into the HPA1 sites of pMSCV-PURO. Clones were selected by ampicillin resistance, tested for inserts by restriction digest with EcoR1 and Xho1, and sequenced (Functional Genomics). Control (i.e., pMSCV-IRES-GFP) and LCN7 retroviral super- natants were produced and infected into murine brain microvas- cular MB114 endothelial cells and SVEC tumor derived endothelial cells as described previously [36,37,38,39]. Infected cells were isolated by puromycin selections (4 mg/ml) to yield stable polyclonal populations of control or LCN7-overexpressing cells. LCN7 expression was confirmed by TCA/DOC precipita- tion of conditioned media with anti-FLAG M2 monoclonal antibodies. Reverse-Transcription PCR Total RNA was collected from either zebrafish embryos or from mammalian endothelial cells using Tri Reagent (Sigma) according to the manufacturer’s recommendations. cDNA was generated from 1 mg of total RNA that was reverse transcribed in 10 ml reactions according to the manufacturer’s recommendations (iScript, Biorad), diluted to 100 ml, and used for PCR (2.5 ml cDNA/25 ml reaction). Oligonucleotides used to amplify zebrafish LCN7 were as follows: Forward: 59 TGCCTTACCTGGACACCATCTGTTA, Reverse: 59 ACCGGTGGCCATTTCTTTCACAAG. Oligonucleotides used to amplify zebrafish GAPDH were as follows. 59 AGGCTTCTCA- CAAACGAGGACACA, Reverse 59 ATCAATGACCAGTTTGC- CGCCTTC. Oligonucleotides used to amplify mouse LCN7 were as follows. 59 TGCCTTACCTGGACACCATCTGTTA, reverse 59 ACCGGTGGCCATTTCTTTCACAAG. Oligonucleotides used to amplify mouse GAPDH were as follows. 59 GACAATGAA- TACGGCTACAGCAAC, Reverse 59 GTGCAGCGAACTTTA- TTGATGGTA. All RT-PCR products were sequenced at least once to confirm amplification of correct target sequences. Author Contributions Conceived and designed the experiments: ARA. Performed the experi- ments: LJB MBA MC TR AS ARA. Analyzed the data: LJB MBA TT ARA. Contributed reagents/materials/analysis tools: ARA. Wrote the paper: LJB ARA. 10. Kobayashi M, Kinouchi T, Hakamata Y, Kamiakito T, Kuriki K, et al. (2001) Isolation of an androgen-inducible novel lipocalin gene, Arg1, from androgen- dependent mouse mammary Shionogi carcinoma cells. J Steroid Biochem Mol Biol 77: 109–115. 11. Helton R, Cui J, Scheel JR, Ellison JA, Ames C, et al. (2005) Brain-specific knock-out of hypoxia-inducible factor-1alpha reduces rather than increases hypoxic-ischemic damage. J Neurosci 25: 4099–4107. 12. 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(2003) An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells. J Biol Chem 278: 17084–17092. 17. Folkman J, Shing Y (1992) Angiogenesis. J Biol Chem 267: 10931–10934. PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e13905