472 | wileyonlinelibrary.com/journal/jai J Appl Ichthyol. 2018;34:472–480. © 2018 Blackwell Verlag GmbH 1 | INTRODUCTION In 2008, a new vitamin K-dependent protein was identified and entitled Gla-rich protein (GRP, also known as UCMA – Upper zone of growth plate and Cartilage Matrix Associated protein) due to its high Gla content in the mature form. This protein was first identified and isolated from sturgeon by our group and found to have orthologs in all taxonomic groups of vertebrates and a paralog (Grp2, also known as Ucmaa) in bony fish (Fazenda, Silva, Cancela, & Conceição, 2012; Viegas et al., 2008). Initially, GRP was described as being specific to cartilage (Surmann-Schmitt et al., 2008; Tagariello et al., 2008), but also since been detected in blood cells, skin, vascular system, and in osteo- cytes of bone, being also associated to ectopic calcifications in dif- ferent human pathologies (Viegas et al., 2009). In addition to the presence of Gla residues, no other identifiable functional domains were described. The association of this protein with high affinity calcium binding (Viegas et al., 2008) and accumulation at sites of pathological calcification (Viegas et al., 2009) suggests that GRP may function as a modulator of calcium availability. Additionally, its association with fibrillary collagens type II, V, IX, and XI, but not type I collagen, also suggests that GRP may be involved in the organiza- tion and/or stabilization of cartilage matrix and may play a key role in chondrogenesis (revised in Cancela, Conceição, & Laizé, 2012). Based on its negative action in the differentiation of preosteoblast MC3T3-E1 cell line and murine primary calvaria cells, GRP has been proposed as functioning as an inhibitor of osteogenesis (Surmann- Schmitt et al., 2008). Considering the many studies available, GRP might have a physiological role in cartilage development and calci- fication during skeleton formation (revised in Cancela et al., 2012). However, no clear results are available to date concerning its func- tion. While in mice the knockout did not appear to have a specific phenotype (Eitzinger et al., 2012), the morpholino-mediated knock- down of grp1 (also known as ucmab) in zebrafish showed a weak and transient phenotype affecting cartilage development (slight overall growth retardation, smaller head, reduced eye pigmentation, irreg- ular and undulating notochord), but also severe and definitive phe- notypes where morphants lacked craniofacial cartilage in almost all cases and with their notochord strongly spirally compressed (Neacsu et al., 2011). Due to the apparent differences between mouse GRP Received: 19 January 2016 | Accepted: 2 August 2017 DOI: 10.1111/jai.13662 ORIGINAL ARTICLE Generation of zebrafish Danio rerio (Hamilton, 1822) transgenic lines overexpressing a heat-shock mediated Gla-rich protein C. Fazenda 1,2 | G. Martins 1 | P. J. Gavaia 1,3 | M. L. Cancela 1,3,4 | N. Conceição 1,3,4 1 Centre of Marine Sciences (CCMAR), University of Algarve, Faro, Portugal 2 PhD program in Biomedical Sciences, Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal 3 Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal 4 Algarve Biomedical Center, University of Algarve, Faro, Portugal Correspondence Natércia Conceição, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Faro, Portugal. Email: nconcei@ualg.pt Funding information FCT - Foundation for Science and Technology through project UID/ Multi/04326/2013, Grant/Award Number: SFRH/BD/66745/2009 and SFRH/ BPD/111898/2015 Summary In an effort to provide more information on the function of Grp isoforms in zebrafish, two zebrafish transgenic lines were generated that could conditionally overexpress grp genes. Reported here is the process and strategy used for the generation of these transgenic lines. Overexpression of Grp1 and Grp2 were induced by heat-shock treatment to investigate their possible roles in the skeleton development. Differences in skeleton development were analysed by alizarin red staining in the larvae. Although no differences in total deformities between wild type and Grp overexpressing larvae were observed, Grp1 and Grp2 overexpressing larvae presented a high percentage of deformities in abdominal and caudal fin vertebra, respectively, with a suggested spe- cific localization of deformities for each isoform.