A transgenic zebrafish model of neutrophilic inflammation

Abstract

We have established an in vivo model for genetic analysis of the inflammatory response by generating a transgenic zebrafish line that expresses GFP under the neutrophil-specific myeloperoxidase promoter. We show that inflammation is induced after transection of the tail of zebrafish larvae and that this inflammation subsequently resolves over a similar time course to mammalian systems. Quantitative data can be generated from this model by counting of fluorescent cells or by digital image analysis. In addition, we show that the resolution of experimentally induced inflammation can be inhibited by the addition of a pancaspase inhibitor, zVD.fmk, demonstrating that experimental manipulation of the resolution of inflammation is possible in this model. IntroductionNeutrophilic inflammation is essential for the maintenance of health and life, but failure to resolve the response in a timely manner can cause irreparable tissue damage because of the release of toxic granule contents of persisting neutrophils. 1 An understanding of the genetic basis of inflammation resolution would undoubtedly provide an important basis for the development of approaches to limiting neutrophil-mediated tissue injury. To facilitate such a genetic analysis, an animal model in which the cellular components of inflammation can be readily visualized in wild-type and genetically manipulated individuals is required.Zebrafish larvae are transparent, allowing excellent visualization of fluorescent proteins in cellular processes in vivo. Zebrafish neutrophils (heterophils) are identifiable from approximately 48 hours after fertilization, 2 and the innate immune system exists in isolation from any adaptive system, which does not arise until approximately 4 weeks after fertilization. 3 A range of tools is available for the genetic manipulation of the zebrafish, as are extensive genomics resources, including a draft sequence of the entire genome. We therefore selected this species as an ideal organism for the generation of a simplified, genetically tractable model using fluorescent neutrophils to track the inflammatory response. Here, we report the generation of a transgenic zebrafish line for use in such experiments and describe the onset and resolution of inflammation in this model. We show that inflammation proceeds with kinetics similar to those in mammalian systems and that experimental manipulation of inflammation in this system is achievable and quantifiable. Materials and methodsZebrafish were maintained according to standard protocols. 4 Reagents were from Sigma (Poole, United Kingdom) unless otherwise specified. zVD.fmk was from Bachem (Weil am Rhein, Germany). MPO::GFP lineBAC (zC91B8) was modified by the use of a red recombinase system in EL250 cells (gift of Dr Neal Copeland, National Cancer Institute, Frederick, MD). 5 EGFP with an SV40 polyadenylation site (Clontech, Palo Alto, CA) was inserted at the mpo (also called mpx) ATG start site. This BAC, linearized with PI-Sce1, was used to generate stable transgenic lines according to publis...

Ribosomal biogenesis genes play an essential and p53-independent role in zebrafish pancreas development

“…S2A-C,E,F). As in other species, zebrafish neutrophils are dynamically recruited to sites of injury (Mathias et al, 2006;Renshaw et al, 2006). Even when Sbds ATG MO-injected embryos were subjected to epithelial injury, no recruitment of mpo-positive neutrophils was observed (supplementary material Fig.…”

Regard à travers le danio pour mieux comprendre les interactions hôte/pathogène

“…De nombreuses lignées transgéniques permettent également d'induire l'expression d'une protéine d'intérêt sous le contrôle d'un promoteur spécifique d'un tissu ou d'un type cellulaire. Ainsi, les lignées exprimant différents fluorochromes sous le contrôle de promoteurs spécifiques des macrophages ou des polynucléaires neutrophiles sont particulièrement remarquables pour l'étude des interactions hôte-pathogène in vivo [12,13]. En exploitant la transparence des embryons, l'utilisation de bactéries fluorescentes, combinée aux lignées transgéniques, permet de déchiffrer avec une grande précision les interactions hôte-pathogène à l'échelle cellulaire (Figure 2).…”

Immunological tools: Engaging students in the use and analysis of flow cytometry and enzyme‐linked immunosorbent assay (ELISA)

“…The first laboratory exercise, which was performed during laboratory sessions 1 and 2, required students to identify the cell type expressing enhanced green fluorescent protein (EGFP) in a transgenic zebrafish line by comparing forward [25][26][27][28][29][30].…”