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Zebrafish Models for Human Genetic Disease Studies

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A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Human Genomics and Genetic Diseases".

Deadline for manuscript submissions: closed (25 May 2023) | Viewed by 26420

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Special Issue Editors

Dr. Jason N. Berman

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Guest Editor

Department of Pediatrics, University of Ottawa, 451 Smyth Road, Room #3206, Ottawa, ON K1H 8M5, Canada
Interests: zebrafish; genetics; disease model; genome editing; mutation; genetic diseases; drug screening; CRISPR/Cas9

Dr. Sergey Prykhozhij

E-Mail Website
Guest Editor

CHEO Research Institute, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada
Interests: zebrafish; disease models; rare genetic diseases; cancer research; p53; CRISPR/Cas9; gene editing; muscular dystrophy; hematopoiesis; bioinformatics

Special Issue Information

Dear Colleagues,

The zebrafish continues to fascinate the research community and the public at large for its versatility as a model system, enabling researchers to answer key biological questions at a whole-organism level. Equally important is the field of disease modeling in zebrafish, which involves in-depth understanding of homologous genetic changes corresponding to specific human genetic diseases with an aim to identify new treatments. Such disease model investigations may lead to novel insights that will guide new avenues for therapeutics development.

In addition to classical approaches, recent technological advances in genome editing and sequencing, synthesis of RNA and DNA molecules, chemical screening, imaging methods, bioinformatics, and machine learning have enabled many improvements in how zebrafish disease modeling is conducted, resulting in more robust, representative, and predictive preclinical models. The current successes in this area make it even more exciting to look forward to new disease models and tools to investigate them. Ultimately, this work in zebrafish-based disease modeling should improve clinical outcomes in patients suffering from these diseases by identifying biomarkers, informing gene therapy methods, or developing pharmacological leads.

We invite zebrafish researchers working on any aspect of disease modeling in zebrafish to contribute their original articles, short communications, and reviews. This Special Issue seeks to attract either basic studies of disease-linked genes in zebrafish or more translational studies of zebrafish disease models, as well as studies describing novel experimental or computational tools to investigate these models and reviews on disease modeling topics.

Dr. Jason N. Berman
Dr. Sergey Prykhozhij
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

zebrafish
genetics
disease model
genome editing
mutation
genetic diseases
drug screening
CRISPR/Cas9

Published Papers (10 papers)

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Research

Jump to: Review

23 pages, 4006 KiB

Open AccessArticle

POPDC1 Variants Cause Atrioventricular Node Dysfunction and Arrhythmogenic Changes in Cardiac Electrophysiology and Intracellular Calcium Handling in Zebrafish

by Matthew R. Stoyek, Sarah E. Doane, Shannon E. Dallaire, Zachary D. Long, Jessica M. Ramia, Donovan L. Cassidy-Nolan, Kar-Lai Poon, Thomas Brand and T. Alexander Quinn

Genes 2024, 15(3), 280; https://0-doi-org.brum.beds.ac.uk/10.3390/genes15030280 - 23 Feb 2024

Viewed by 853

Abstract

Popeye domain-containing (POPDC) proteins selectively bind cAMP and mediate cellular responses to sympathetic nervous system (SNS) stimulation. The first discovered human genetic variant (POPDC1^S201F) is associated with atrioventricular (AV) block, which is exacerbated by increased SNS activity. Zebrafish carrying the homologous mutation (popdc1^S191F) display a similar phenotype to humans. To investigate the impact of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling, homozygous popdc1^S191F and popdc1 knock-out (popdc1^KO) zebrafish larvae and adult isolated popdc1^S191F hearts were studied by functional fluorescent analysis. It was found that in popdc1^S191F and popdc1^KO larvae, heart rate (HR), AV delay, action potential (AP) and calcium transient (CaT) upstroke speed, and AP duration were less than in wild-type larvae, whereas CaT duration was greater. SNS stress by β-adrenergic receptor stimulation with isoproterenol increased HR, lengthened AV delay, slowed AP and CaT upstroke speed, and shortened AP and CaT duration, yet did not result in arrhythmias. In adult popdc1^S191F zebrafish hearts, there was a higher incidence of AV block, slower AP upstroke speed, and longer AP duration compared to wild-type hearts, with no differences in CaT. SNS stress increased AV delay and led to further AV block in popdc1^S191F hearts while decreasing AP and CaT duration. Overall, we have revealed that arrhythmogenic effects of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling in zebrafish are varied, but already present in early development, and that AV node dysfunction may underlie SNS-induced arrhythmogenesis associated with popdc1 mutation in adults. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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15 pages, 5992 KiB

Open AccessArticle

lmo4a Contributes to Zebrafish Inner Ear and Vestibular Development via Regulation of the Bmp Pathway

by Le Sun, Lu Ping, Ruzhen Gao, Bo Zhang and Xiaowei Chen

Genes 2023, 14(7), 1371; https://0-doi-org.brum.beds.ac.uk/10.3390/genes14071371 - 28 Jun 2023

Cited by 1 | Viewed by 1147

Abstract

Background: In vertebrates, the development of the inner ear is a delicate process, whereas its relating molecular pathways are still poorly understood. LMO4, an LIM domain-only transcriptional regulator, is drawing an increasing amount of interest for its multiple roles regarding human embryonic development and the modulation of ototoxic side effects of cisplatin including cochlear apoptosis and hearing loss. The aim of the present study is to further explore the role of lmo4a in zebrafish inner ear development and thus explore its functional role. Methods: The Spatial Transcript Omics DataBase was referred to in order to evaluate the expression of lmo4a during the first 24 h of zebrafish development. In situ hybridization was applied to validate and extend the expression profile of lmo4a to 3 days post-fertilization. The morpholino (MO) knockdown and CRISPR/Cas9 knockout (KO) of lmo4a was applied. Morphological analyses of otic vesical, hair cells, statoacoustic ganglion and semicircular canals were conducted. The swimming pattern of lmo4a KO and MO zebrafish was tracked. In situ hybridization was further applied to verify the expression of genes of the related pathways. Rescue of the phenotype was attempted by blockage of the bmp pathway via heat shock and injection of Dorsomorphin. Results: lmo4a is constitutively expressed in the otic placode and otic vesicle during the early stages of zebrafish development. Knockdown and knockout of lmo4a both induced smaller otocysts, less hair cells, immature statoacoustic ganglion and malformed semicircular canals. Abnormal swimming patterns could be observed in both lmo4a MO and KO zebrafish. eya1 in preplacodal ectoderm patterning was downregulated. bmp2 and bmp4 expressions were found to be upregulated and extended in lmo4a morphants, and blockage of the Bmp pathway partially rescued the vestibular defects. Conclusions: We concluded that lmo4a holds a regulative effect on the Bmp pathway and is required for the normal development of zebrafish inner ear. Our study pointed out the conservatism of LMO4 in inner ear development between mammals and zebrafish as well as shed more light on the molecular mechanisms behind it. Further research is needed to distinguish the relationships between lmo4 and the Bmp pathway, which may lead to diagnostic and therapeutic approaches towards human inner ear malformation. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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11 pages, 1620 KiB

Open AccessArticle

phox2ba: The Potential Genetic Link behind the Overlap in the Symptomatology between CHARGE and Central Congenital Hypoventilation Syndromes

by Jessica E. MacLean, Jaime N. Wertman, Sergey V. Prykhozhij, Emily Chedrawe, Stewart Langley, Shelby L. Steele, Kevin Ban, Kim Blake and Jason N. Berman

Genes 2023, 14(5), 1086; https://0-doi-org.brum.beds.ac.uk/10.3390/genes14051086 - 15 May 2023

Viewed by 1274

Abstract

CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7). CHD7 is involved in regulating neural crest development, which gives rise to tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of PHOX2B (paired-like homeobox 2b) underlies CCHS. Employing a chd7-null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of phox2b. Heart rates were lower in chd7 mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that chd7 mutants took longer to become anesthetized, with higher respiratory rates during recovery. chd7 mutant larvae demonstrated unique phox2ba expression patterns. The knockdown of phox2ba reduced larval heart rates similar to chd7 mutants. chd7 mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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16 pages, 2514 KiB

Open AccessArticle

Cytoskeletal Keratins Are Overexpressed in a Zebrafish Model of Idiopathic Scoliosis

by Melissa Cuevas, Elizabeth Terhune, Cambria Wethey, MkpoutoAbasi James, Rahwa Netsanet, Denisa Grofova, Anna Monley and Nancy Hadley Miller

Genes 2023, 14(5), 1058; https://0-doi-org.brum.beds.ac.uk/10.3390/genes14051058 - 09 May 2023

Viewed by 1659

Abstract

Idiopathic scoliosis (IS) is a three-dimensional rotation of the spine >10 degrees with an unknown etiology. Our laboratory established a late-onset IS model in zebrafish (Danio rerio) containing a deletion in kif7. A total of 25% of kif7^co^63/co63 zebrafish develop spinal curvatures and are otherwise developmentally normal, although the molecular mechanisms underlying the scoliosis are unknown. To define transcripts associated with scoliosis in this model, we performed bulk mRNA sequencing on 6 weeks past fertilization (wpf) kif7^co^63/co63 zebrafish with and without scoliosis. Additionally, we sequenced kif7^co^63/co63, kif7^co^63/+, and AB zebrafish (n = 3 per genotype). Sequencing reads were aligned to the GRCz11 genome and FPKM values were calculated. Differences between groups were calculated for each transcript by the t-test. Principal component analysis showed that transcriptomes clustered by sample age and genotype. kif7 mRNA was mildly reduced in both homozygous and heterozygous zebrafish compared to AB. Sonic hedgehog target genes were upregulated in kif7^co^63/co63 zebrafish over AB, but no difference was detected between scoliotic and non-scoliotic mutants. The top upregulated genes in scoliotic zebrafish were cytoskeletal keratins. Pankeratin staining of 6 wpf scoliotic and non-scoliotic kif7^co^63/co63 zebrafish showed increased keratin levels within the zebrafish musculature and intervertebral disc (IVD). Keratins are major components of the embryonic notochord, and aberrant keratin expression has been associated with intervertebral disc degeneration (IVDD) in both zebrafish and humans. The role of increased keratin accumulation as a molecular mechanism associated with the onset of scoliosis warrants further study. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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15 pages, 6799 KiB

Open AccessArticle

vwa1 Knockout in Zebrafish Causes Abnormal Craniofacial Chondrogenesis by Regulating FGF Pathway

by Xiaomin Niu, Fuyu Zhang, Lu Ping, Yibei Wang, Bo Zhang, Jian Wang and Xiaowei Chen

Genes 2023, 14(4), 838; https://0-doi-org.brum.beds.ac.uk/10.3390/genes14040838 - 30 Mar 2023

Viewed by 1462

Abstract

Hemifacial microsomia (HFM), a rare disorder of first- and second-pharyngeal arch development, has been linked to a point mutation in VWA1 (von Willebrand factor A domain containing 1), encoding the protein WARP in a five-generation pedigree. However, how the VWA1 mutation relates to the pathogenesis of HFM is largely unknown. Here, we sought to elucidate the effects of the VWA1 mutation at the molecular level by generating a vwa1-knockout zebrafish line using CRISPR/Cas9. Mutants and crispants showed cartilage dysmorphologies, including hypoplastic Meckel’s cartilage and palatoquadrate cartilage, malformed ceratohyal with widened angle, and deformed or absent ceratobranchial cartilages. Chondrocytes exhibited a smaller size and aspect ratio and were aligned irregularly. In situ hybridization and RT-qPCR showed a decrease in barx1 and col2a1a expression, indicating abnormal cranial neural crest cell (CNCC) condensation and differentiation. CNCC proliferation and survival were also impaired in the mutants. Expression of FGF pathway components, including fgf8a, fgfr1, fgfr2, fgfr3, fgfr4, and runx2a, was decreased, implying a role for VWA1 in regulating FGF signaling. Our results demonstrate that VWA1 is essential for zebrafish chondrogenesis through effects on condensation, differentiation, proliferation, and apoptosis of CNCCs, and likely impacts chondrogenesis through regulation of the FGF pathway. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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16 pages, 5781 KiB

Open AccessArticle

Loss of calpain3b in Zebrafish, a Model of Limb-Girdle Muscular Dystrophy, Increases Susceptibility to Muscle Defects Due to Elevated Muscle Activity

by Sergey V. Prykhozhij, Lucia Caceres, Kevin Ban, Anna Cordeiro-Santanach, Kanneboyina Nagaraju, Eric P. Hoffman and Jason N. Berman

Genes 2023, 14(2), 492; https://0-doi-org.brum.beds.ac.uk/10.3390/genes14020492 - 15 Feb 2023

Cited by 1 | Viewed by 2604

Abstract

Limb-Girdle Muscular Dystrophy Type R1 (LGMDR1; formerly LGMD2A), characterized by progressive hip and shoulder muscle weakness, is caused by mutations in CAPN3. In zebrafish, capn3b mediates Def-dependent degradation of p53 in the liver and intestines. We show that capn3b is expressed in the muscle. To model LGMDR1 in zebrafish, we generated three deletion mutants in capn3b and a positive-control dmd mutant (Duchenne muscular dystrophy). Two partial deletion mutants showed transcript-level reduction, whereas the RNA-less mutant lacked capn3b mRNA. All capn3b homozygous mutants were developmentally-normal adult-viable animals. Mutants in dmd were homozygous-lethal. Bathing wild-type and capn3b mutants in 0.8% methylcellulose (MC) for 3 days beginning 2 days post-fertilization resulted in significantly pronounced (20–30%) birefringence-detectable muscle abnormalities in capn3b mutant embryos. Evans Blue staining for sarcolemma integrity loss was strongly positive in dmd homozygotes, negative in wild-type embryos, and negative in MC-treated capn3b mutants, suggesting membrane instability is not a primary muscle pathology determinant. Increased birefringence-detected muscle abnormalities in capn3b mutants compared to wild-type animals were observed following induced hypertonia by exposure to cholinesterase inhibitor, azinphos-methyl, reinforcing the MC results. These mutant fish represent a novel tractable model for studying the mechanisms underlying muscle repair and remodeling, and as a preclinical tool for whole-animal therapeutics and behavioral screening in LGMDR1. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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17 pages, 3578 KiB

Open AccessArticle

Uricase-Deficient Larval Zebrafish with Elevated Urate Levels Demonstrate Suppressed Acute Inflammatory Response to Monosodium Urate Crystals and Prolonged Crystal Persistence

by Tanja Linnerz, Yih Jian Sung, Leah Rolland, Jonathan W. Astin, Nicola Dalbeth and Christopher J. Hall

Genes 2022, 13(12), 2179; https://0-doi-org.brum.beds.ac.uk/10.3390/genes13122179 - 22 Nov 2022

Cited by 3 | Viewed by 1690

Abstract

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures (‘tophi’) composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient ‘hyperuricemic’ larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox^−/− larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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Graphical abstract

16 pages, 3683 KiB

Open AccessFeature PaperArticle

Mutation of foxl1 Results in Reduced Cartilage Markers in a Zebrafish Model of Otosclerosis

by Alexia Hawkey-Noble, Justin A. Pater, Roshni Kollipara, Meriel Fitzgerald, Alexandre S. Maekawa, Christopher S. Kovacs, Terry-Lynn Young and Curtis R. French

Genes 2022, 13(7), 1107; https://0-doi-org.brum.beds.ac.uk/10.3390/genes13071107 - 21 Jun 2022

Cited by 4 | Viewed by 2590

Abstract

Bone diseases such as otosclerosis (conductive hearing loss) and osteoporosis (low bone mineral density) can result from the abnormal expression of genes that regulate cartilage and bone development. The forkhead box transcription factor FOXL1 has been identified as the causative gene in a family with autosomal dominant otosclerosis and has been reported as a candidate gene in GWAS meta-analyses for osteoporosis. This potentially indicates a novel role for foxl1 in chondrogenesis, osteogenesis, and bone remodelling. We created a foxl1 mutant zebrafish strain as a model for otosclerosis and osteoporosis and examined jaw bones that are homologous to the mammalian middle ear bones, and mineralization of the axial skeleton. We demonstrate that foxl1 regulates the expression of collagen genes such as collagen type 1 alpha 1a and collagen type 11 alpha 2, and results in a delay in jawbone mineralization, while the axial skeleton remains unchanged. foxl1 may also act with other forkhead genes such as foxc1a, as loss of foxl1 in a foxc1a mutant background increases the severity of jaw calcification phenotypes when compared to each mutant alone. Our zebrafish model demonstrates atypical cartilage formation and mineralization in the zebrafish craniofacial skeleton in foxl1 mutants and demonstrates that aberrant collagen expression may underlie the development of otosclerosis. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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20 pages, 4053 KiB

Open AccessArticle

A New Zebrafish Model to Measure Neuronal α-Synuclein Clearance In Vivo

by Ana Lopez, Alena Gorb, Nuno Palha, Angeleen Fleming and David C. Rubinsztein

Genes 2022, 13(5), 868; https://0-doi-org.brum.beds.ac.uk/10.3390/genes13050868 - 12 May 2022

Cited by 7 | Viewed by 7492

Abstract

The accumulation and aggregation of α-synuclein (α-SYN) is a common characteristic of synucleinopathies, such as Parkinson’s Disease (PD), Dementia with Lewy Bodies (DLB) or Multiple System Atrophy (MSA). Multiplications of the wildtype gene of α-SYN (SNCA) and most point mutations make α-SYN more aggregate-prone, and are associated with mitochondrial defects, trafficking obstruction, and impaired proteostasis, which contribute to elevated neuronal death. Here, we present new zebrafish models expressing either human wildtype (wt), or A53T mutant, α-SYN that recapitulate the above-mentioned hallmarks of synucleinopathies. The appropriate clearance of toxic α-SYN has been previously shown to play a key role in maintaining cell homeostasis and survival. However, the paucity of models to investigate α-SYN degradation in vivo limits our understanding of this process. Based on our recently described imaging method for measuring tau protein clearance in neurons in living zebrafish, we fused human SNCA to the photoconvertible protein Dendra2 which enabled analyses of wt and A53T α-SYN clearance kinetics in vivo. Moreover, these zebrafish models can be used to investigate the kinetics of α-SYN aggregation and to study the mechanisms, and potential new targets, controlling the clearance of both soluble and aggregated α-SYN. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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Review

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16 pages, 1251 KiB

Open AccessReview

A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases

by Blake Carrington, Kevin Bishop and Raman Sood

Genes 2022, 13(5), 857; https://0-doi-org.brum.beds.ac.uk/10.3390/genes13050857 - 11 May 2022

Cited by 4 | Viewed by 3722

Abstract

The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects. Full article

(This article belongs to the Special Issue Zebrafish Models for Human Genetic Disease Studies)

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