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Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune hoemostasis, and recognition of microbial pahtogens. Galectins are β-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.
The zebrafish (Danio rerio) is a widely used model organism to study a broad spectrum of human normal and pathological processes, including development, autoimmune, neoplastic and infectious disease (1–3). The growing interest in using zebrafish as a genetically tractable model system is due to its multiple advantages relative to the mammalian models, which include high fecundity, external fertilization, rapid development, transparent embryo, low maintenance cost, easy genetic manipulation and an extensive collection of mutants currently available (4–6). Zebrafish and human genomes have been shown to share highly conserved structural and functional features (7). Hundreds of zebrafish genes have been identified and its full genome sequence is now available online (http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/). Moreover, D. rerio exhibits some higher-level behaviors previously observed only in mammals, such as memory, conditioned responses and schooling (8,9). In recent years, the zebrafish model has proven useful to gain new insight into the functional aspects of protein-carbohydrate interactions, such as those mediated by galectins (10–12). Galectins are β-galactosyl-binding lectins, which share primary structural homology in their carbohydrate-recognition domains (CRDs) (13). They are classified into three major structural types: (i) proto-type galectins, which contain one carbohydrate recognition domain (CRD) and form homodimers; (ii) chimera-type galectins, which have a single CRD and can oligomerize forming trimers and pentamers; (iii) tandem-repeat-type galectins, which are comprised of two CRD joined by a linker peptide (14). Galectins participate in a multitude of biological processes, such as development, apoptosis, tumor metastasis and regulation of immune responses (15–19). All three structural types of galectins have been identified and characterized in various tissues, plasma and mucus of teleost fish, such as zebrafish (12,20–23). Among the various methodologies for elucidating the biological role(s) of a particular protein of interest, disruption of gene expression represents a useful approach. Multiple strategies have been developed to modulate gene expression at a genetic or epigenetic level (Table 1). Several of these methodologies have been recently applied to functional studies of galectins (10,24,25).
Morpholinos (MOs) are antisense oligonucleotides derivatized with morpholine rings to increase stability, and designed to anneal close to the start codon of the selected gene and disrupt its translation, or to the splice acceptor sequence to induce incorrectly spliced mRNA (26). The MOs do not degrade their mRNA targets, but (i) block mRNA translation by targeting the 5'-UTR through the first 25 bases of coding sequence (10), or (ii) alter the translation modifying pre-mRNA processing by targeting splice junctions or regulatory sites (Fig 1) (27,28). Thus, the “morphant” phenotype results from disrupted protein expression levels. Although their effects are only transient, MOs are relatively long-lived inside the cell upon delivery, with the effect on pre-mRNA splicing or translation lasting for up to five days following microinjection. This technique allows for the rapid manipulation and interrogation of complex processes such as embryonic development, organ formation, innate immunity, and host-pathogen interactions (3,10,28). However, since the effects of mRNA suppression by MO oligos are temporary and not inheritable, and there is no suitable way to deliver them systemically in adult fish, this system is not suitable for more integrated approaches. For inheritable genetic modulation, a set of other techniques has been introduced in the last 15 years (Table 1).
CRISPR-Cas is a genome editing approach based on the prokaryotic immune system. By using a segment of virus-derived DNA from its CRISPR array and processing to crRNA targeting the viral genome, the system leads to the inactivation or degradation of targeted DNA by the CAS-crRNA complex (29). Cas9 nuclease transgenically expressed in vertebrates is active and able to cleave target DNA when directed by a short guide RNA (gRNA), which at its 5'-end contains 20 base pairs of complementary target DNA (30–32).
Here we describe in detail the use of MOs and CRISPR-Cas to modulate galectin expression in the zebrafish model to elucidate their functions in development and immunity. The MOs Drgal1-L1-MO and Drgal1-L2-MO were designed to block translation initiation based on 5'-UTR sequences of Drgal1-L1 and Drgal1-L2, respectively (Table 2). MOs are usually validated by in vitro blockade of the corresponding protein expression using the TNT SP6 Coupled Rabbit Reticulocyte Lysate System. Following MO-oligo injection of zebrafish embryos, expression of the corresponding protein is inspected by whole mount antibody staining. Potential phenotypes are inspected under microscope and by whole mount antibody staining of a specific marker. Zebrafish embryos injected with validated Drgal1-L2-MO (Fig. 2A) show dramatically reduced Drgal1-L2 expression as observed by whole mount antibody staining (Fig. 2C). Drgal1-L2 is strongly expressed in the notochord during early embryogenesis and Drgal1-L2 knock-down results in a characteristic phenotype with a short and bent tail (Fig. 2C). Microscopically, the phenotype exhibits disrupted muscle fiber organization as observed by whole mount immunostaining with the F59 antibody (monoclonal anti-myosin antibody), a marker for slow muscle (Fig. 2E).
MOs for each gene are designed based on the gene sequence to obtain translational blocker (designed to bind close to start codon disrupting the translation) or splice blocker (designed to bind to splice acceptor sequence to induce incorrectly spliced mRNA). For example, Drgal1-L1-MO and Drgal1-L2-MO shown in Table 2 are translational blockers of the Drgal1-L1 and Drgal1-2, respectively. These MOs were custom synthesized by Gene Tools (www.gene-tools.com) (Note a).
Experimental work described here was supported by grant 5R01GM070589-06 from the National Institutes of Health to GRV.