Isolation of Sea Bass Erythroferrone Gene

Pairs of oligonucleotide PCR primers were designed according to conserved regions of erfe mRNA sequences from other fish and mammalian species, available in the National Center for Biotechnology Information nucleotide database (http://www.ncbi.nlm.nih.gov/) and Ensembl (http://www.ensembl.org/) and cDNA preparations from spleen and kidney were used in PCR amplifications (22, 23). PCR products were run on 1.2% agarose gels, relevant fragments purified with the NZYGelpure kit (NZYtech, Lisbon, Portugal), cloned into pCR™2.1-TOPO^® vectors, propagated in One Shot^® Mach1™-T1R competent cells (Invitrogen, Life Technologies, Carlsbad, CA) and sent for sequencing (Eurofins Genomics, Ebersberg, Germany). Both strands were sequenced, and chromatograms were analyzed in FinchTV (Geospiza, Seattle WA, USA) and assembled using Multalin (http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html).