This article provides recommendations for the care of laboratory zebrafish ( Danio rerio) as part of the further implementation of Annex A to the European Convention on the protection of vertebrate animals used for experimental and other scientific purposes, EU Commission Recommendation 2007/526/EC and the fulfilment of Article 33 of EU Directive 2010/63, both concerning the housing and care of experimental animals. The recommendations provide guidance on best practices and ranges of husbandry parameters within which zebrafish welfare, as well as reproducibility of experimental procedures, are assured. Husbandry procedures found today in zebrafish facilities are numerous. While the vast majority of these practices are perfectly acceptable in terms of zebrafish physiology and welfare, the reproducibility of experimental results could be improved by further standardisation of husbandry procedures and exchange of husbandry information between laboratories. Standardisation protocols providing ranges of husbandry parameters are likely to be more successful and appropriate than the implementation of a set of fixed guidance values neglecting the empirically successful daily routines of many facilities and will better reflect the wide range of environmental parameters that characterise the natural habitats occupied by zebrafish. A joint working group on zebrafish housing and husbandry recommendations, with members of the European Society for Fish Models in Biology and Medicine (EUFishBioMed) and of the Federation of European Laboratory Animal Science Associations (FELASA) has been given a mandate to provide guidelines based on a FELASA list of parameters, ‘Terms of Reference’.

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (-37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.Prolactin (PRL) is a pituitary polypeptide hormone that is implicated in many physiological actions in vertebrates including fish (1). Since the pioneering work by Pickford and Phillips in 1959 (2) demonstrating that hypophysectomized Fundulus heteroclitus require PRL for survival in freshwater, numerous studies have confirmed that PRL is one of the major hormones regulating the maintenance of water and electrolyte homeostasis on osmoregulatory surfaces (3-5). In tilapia, two distinct PRL forms have been well characterized (tiPRL188 or tiPRL, and tiPRL177 or tiPRLII), which share only 69% identity (6-8).These two tilapia PRLs (tiPRLs) were shown to be differentially regulated during adaptation to a hyperosmotic environment (9-11) and to exhibit both common and distinct biological effects and potencies (6, 10, 12). These effects are mediated by a specific cell membrane receptor. In tilapia, initial studies using ovine PRL (oPRL) as a ligand revealed the presence of PRL receptors in various tissues (13-15). Using bioactive recombinant tiPRL forms (16), high specific binding of PRL (up to 45% with tiPRL188) has recently been shown in gill and kidney, involving only one class of receptors, which binds tiPRL188 with higher affinity than tiPRL177 (17).PRL receptor cDNAs have been cloned from several mammalian species and sources (18)(19)(20) and two avian species (21, 22). These receptors belong to a superfamily including the receptors for growth hormone (GH), cytokines, and erythropoietin (18). This superfamily has several common structural features (23) including two pairs of conserved extracellular cysteine residues, a single transmembrane domain, and an intracellular proline-rich region (24). In lower vertebrates, however, no PRL receptor cDNA has been identified.We report in this paper the isolation of a tiPRL receptor cDNAI by an expression cloning approach (25). The characterized tiPRL receptor is a mature protein of 606 amino acids with a single extracellular unit and a long cytoplasmic domain. Moreover, tissue distribution studies indica...

The gonadal soma-derived factor (GSDF) is a new member of the transforming growth factor beta (TGF-beta) superfamily that regulates the proliferation of the primordial germ cells (PGC) in developing embryos and spermatogonia in juvenile male trout. The gsdf transcripts are expressed in the somatic cells supporting germ cell development. In zebrafish, we show that GSDF is encoded by a single copy gene that generates polymorphic transcripts and proteins. We determined that gsdf gene expression occurs before gonadal differentiation and is restricted to the gonads. Gene expression is maintained in adult granulosa cells and Sertoli cells but decreases in the cells that are in contact with meiotic and postmeiotic germ cells. Using zebrafish transgenic lines, we demonstrate that the 2-kb proximal promoter region of the gsdf gene targets high levels of transgene expression in the Sertoli and granulosa cells, and is sufficient to mimic the temporal expression pattern of the endogenous gsdf gene from 16 days postfertilization onward. We identified within the first 500 bp evolutionarily conserved DNA motifs that may be involved in Sertoli and granulosa cell-specific expression. However, the 2-kb proximal promoter region failed to drive efficient expression of the transgene in the gonads in four transgenic medaka lines. We propose that the proximal promoter region can be used to target candidate gene deregulation in zebrafish granulosa and Sertoli cells. Furthermore, the green fluorescent protein-expressing zebrafish lines produced in the present study are new valuable models for cell lineage tracing during sex differentiation and gametogenesis.

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Plasmacytoid dendritic cells (pDCs) play a crucial role in antiviral innate immunity through their unique capacity to produce large amounts of type I interferons (IFNs) upon viral detection. Tripartite motif (TRIM) proteins have recently come forth as important modulators of innate signaling, but their involvement in pDCs has not been investigated. Here, we performed a rationally streamlined small interfering RNA (siRNA)-based screen of TRIM proteins in human primary pDCs to identify those that are critical for the IFN response. Among candidate hits, TRIM8 emerged as an essential regulator of IFN regulatory factor 7 (IRF7) function. Mechanistically, TRIM8 protects phosphorylated IRF7 (pIRF7) from proteasomal degradation in an E3 ubiquitin ligase-independent manner by preventing its recognition by the peptidyl-prolyl isomerase Pin1. Our findings uncover a previously unknown regulatory mechanism of type I IFN production in pDCs by which TRIM8 and Pin1 oppositely regulate the stability of pIRF7.

A soluble protein that specifically bound 125 I-human growth hormone (hGH) was identified in rainbow trout plasma, using HPLC-gel filtration. The binding affinity of the protein for hGH was 1.2 ؋ 10 9 M ؊1 . 125 I-rainbow trout GH (tGH) was also able to bind to the protein albeit with a lower affinity (6.6 ؋ 10 7 M ؊1 ) than hGH. Crosslinking experiments using 125 I-hGH revealed two specific bands of 150 and 130 kDa. The complex 125 I-hGH-BP could be precipitated by a monoclonal anti-GH receptor antibody, suggesting a close relationship between the plasma GH-BP and the GH receptor. A fourfold increase in the hGH binding to the GH-BP was shown 48 h after transfer of the fishes from freshwater to seawater. The increase in binding was related to a high binding capacity without significant changes in binding affinity. These results suggest a potential role of this related GH-BP as an index of GH effects during seawater adaptation in salmonids.1998 Academic Press