Zebrafish morpholino gene knockdown creative biogene. You can inject mrna or dna of the wildtype gene or a constitutively. Rna interference, rnai, danio rerio, lamin a, lamin b2. This volume presents a historical perspective on morpholinos, an overview of good morpholino practices, techniques for controlling morpholino activity with light, techniques for modulating microrna activity in zebrafish embryos, probing genes during fin regeneration, methods for determining to structure of gene networks during development, electroporation, bacterial knockdowns, pretargeting, diagnostic. Download protocols, publications and useful documents gene. The knockdown phenotype is often more extreme than the mutant phenotype.
This study provides additional proof of concept that zebrafish can be used as an efficient alternative. The discovery of rnai, an evolutionary conserved, innate and potent method of sequencespecific gene knockdown, has therefore opened up a new potential. Jun 12, 2020 in 2015, using zebrafish knockdown and knockout models, stainiers laboratory discovered a novel mechanism of genetic robustness that is the genetic compensation response gcr. Specific rnai mediated gene knockdown in zebrafish cell.
The usefulness of the zebrafish as a model organism originates in its unique combination of genetic and embryological characteristics. Gene knockdown in embryos and the production of transgenic strains are now standard techniques being used to assess physiology. Effective targeted gene knockdown in zebrafish mayo clinic. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted knockdown technology in this model vertebrate.
In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 25day postfertilization zebrafish embryos after morpholinoantisense oligonucleotide knockdown of the candidate gene adp ribosylation factor6 arf6, moarf6. At present the most widely used method of gene knockdown in zebrafish is morpholinos, antisense oligonucleotides which either block translation of mrnas or interfere with correct splicing of mrna. Effective targeted gene knockdown in zebrafish semantic. Feb 19, 2021 the standard approach to block specifically the function of a gene in a zebrafish embryo at the early stages of development is with the modified antisense oligonucleotides called morpholinos 47, 48. Leptina mediates transcription of genes that participate in. Fam60al as a novel factor involved in reprogramming of somatic cell.
The other is that it is a novel igf as proposed by wang et al. To mediate gene specific knockdown in zebrafish, four different mirnas were designed against each of the two zebrafish kcnh2 genes zerg2 and zerg3. You can find zebrafish lines with gene break transposon inserts in zfishbook. An example of an alternate knockdown system would be the introduction of synthetic microrna mirna cassettes into the zebrafish genome to knockdown a desired gene dong et al. Although they have proved a powerful tool, their shortlived nature and numerous reports of offtarget effects has limited their use. The zebrafish danio rerio is a freshwater fish belonging to the minnow family cyprinidae of. Gene knockdown by morpholinomodified oligonucleotides in. Decreased bdnf levels are a major contributor to the. The goals of this project are to determine the zfelovl4 expression patterns in the developing zebrafish and monitor the developmental consequences of zfelovl4 knockdown using morpholino injections. Microinjection of antisense morpholinos, crisprcas9 rnp. If the change in gene expression is caused by an oligonucleotide binding to an mrna or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal dna, and the result is referred to as a transient. Specific rnai mediated gene knockdown in zebrafish cell lines. Knockdown of duplicated zebrafish hoxb1 genes reveals distinct.
Gene knockdown refers to an experimental technique by which the genes are reduced. We analyzed microarray expression data to highlight biological pathways that respond to embryonic zebrafish leptina lepa signaling. Creative biogene can offer professional zebrafish morpholino gene knockdown services. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274 university of oregon, eugene, or 97403 usa. Heritable and lineagespecific gene knockdown in zebrafish embryo. A zebrafish model of human barth syndrome reveals the. Mos can be targeted to block translation of a target mrna, by hybridization to the atg initiation codon, or to prevent correct splicing by annealing to exonintron splice signals in the premrna eisen and smith.
Systems biology analysis was carried out on the data. A beginners guide to understanding and implementing the genetic. If a dna of an organism is genetically modified, the resulting organism is called a knockdown organism. Microinjection of antisense morpholinos, crisprcas9 rnp, and.
Zebrafish rpl10a knockdown and knockout construction. The zebrafish is the most important fish model in developmental andgenetic analyses. The expression of the zebrafish embryonic globin genes, hemoglobin alpha. Knockdown of duplicated zebrafish hoxb1 genes reveals. Replication of a mutant phenotype in a gene knockout inactivation of a gene. To initially test this knockdown strategy, we chose the zebrafish no tail ntl gene 9, 24 as a target to knockdown its expression because homozygous mutant fish lacking this functional gene do not form a tail 25 and this phenotype is easily observable and provides an excellent assay for the disruption of a specific gene function.
Gucy2f zebrafish knockdown a model for gucy2drelated leber. In this chapter, we describe a stepwise protocol of microinjection. Genetic studies in zebrafish have discovered novel genes and pathways in. In addition, for some applications gene knockdown offers distinct advantages as compared to gene knockout approaches. The zebrafish danio rerio is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. Effective heritable gene knockdown in zebrafish using synthetic. Elovl4 expression and morpholino knockdown in zebrafish. Premature termination codonbearing mrna mediates genetic. May 07, 2009 microinjection is a wellestablished and effective method for introducing foreign substances into fertilized zebrafish embryos. Gene knockdown is widely used to study mutant phenotypes of genes for which chemically induced mutations are not available. It is therefore not surprising that the discovery of rnai as an exploitable method of posttranscriptional gene regulation has created a lot of excitement within the zebrafish. Leptina mediates transcription of genes that participate. Morpholino oligomers methods and protocols hong m moulton.
A vast number of genetically engineered zebrafish lines and methods for their. Unfortunately, a heritable and tissuespecific knockdown of gene expression has not yet been developed in animal model organism such as zebrafish, which largely restricts its genetic potential as a vertebrate model of human disorders associated with reduced expression of etiologic genes. As a result, using of zebrafish danio rerio, which is one of the tropical freshwater fish. Fam60al, a coding gene only found in zebrafish and frog among vertebrates. It remains a major challenge to develop conditional gene knockout or effective gene knockdown in adult zebrafish organs. Thus, we have revealed a function shuffling among genes of paralogue group 1. Novel aspects of tdp43s interaction with alsrelated autophagy gene. However, a heritable and tissuespecific knockdown of gene expression has not yet been developed in zebrafish. Rnaimediated gene silencing in zebrafish triggered by. Using this method, antisense morpholinos, crisprcas9 ribonucleoprotein complexes, capped mrna, and dna can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. The zebrafish international resource center zirc is a genetic resource. The major methods for transient gene knockdown morpholino oligonucleotides and stable gene inactivation transcription activator like effector nucleases, talens in zebrafish, described in detail in the text, are depicted schematically. Morpholinos are synthetic molecules, about 25 nucleotide length, and are available at gene tools 49. Its molecular structure contains dna bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups.
Abnormal development of zebrafish after knockout and knockdown. World heritage encyclopedia, the aggregation of the largest online encyclopedias available, and the most definitive collection ever assembled. Targeted gene knockdown in zebrafish using negatively. In order to validate a key gene identified in the analysis, biliary morphogenesis was evaluated in 25day postfertilization zebrafish embryos after morpholinoantisense oligonucleotide knockdown of the candidate gene adp. Pdf in vivo testing of micrornamediated gene knockdown. The utility of zebrafish for physiological research is based on several key features including i a fully sequenced genome, ii rapid 3 month generation times, iii their capacity to produce. Characterization of zebrafish dysferlin by morpholino.
This volume presents a historical perspective on morpholinos, an overview of good morpholino practices, techniques for controlling morpholino activity with light, techniques for modulating microrna activity in zebrafish embryos, probing genes during fin regeneration, methods for determining to structure of gene networks during development. Pdf fluorescentbased methods for gene knockdown and. Although zebrafish is used to model human diseases through mutational and morpholinobased knockdown approaches, there are currently no robust transgenic knockdown tools. Here, we demonstrate a robust microinjection technique for performing mrna overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.
In this system gene knockdown by morpholino oligonucleotides is easily accomplished. However, despite concerted efforts in the field, progress in the use of rnai technologies in zebrafish has been extremely slow and a reliable means of rnaimediated gene knockdown remains elusive. Pectoral fin anomalies in tbx5a knockdown zebrafish. This article is published with open access at abstract a notable advantage of zebrafish as a model this method for knockdown of cardiac troponin t tnnt2a organism is the ease of gene knockdown using morpholino in zebrafish, we could efficiently select the partial tnnt2a antisense oligonucleotide mo. The reduction can occur either through genetic modification or by treatment with a reagent such as a short dna or rna oligonucleotide that has a sequence complementary to either gene or an mrna transcript. This study aimed to assess developmental pectoral fin anomalies in tbx5a morpholino knockdown zebrafish embryos. Here we demonstrate that knockdown of zebrafish photoreceptorspecific gucy2d gene gucy2f, a gene associated with arguably the most prevalent form of lca,1can serve as a readilyavailable, inexpensive animal model for lca1. Properties of gene knockdown system by vectorbased sirna in. Apr 26, 2012 the many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos.
It is all about dosage and validation j invest dermatol. Zebrafish express mammalian set of metazoan pitps plus a unique pitp. For instance, zebrafish with the mutated myo18b gene can serve as a model for. So far, genetic manipulation in zebrafish has been limited to the. In vivo testing of micrornamediated gene knockdown in zebrafish. Microarray expression measures for 26,046 genes were evaluated from lepa morpholino oligonucleotide knockdown, recombinant leptina rescue, and uninjected control zebrafish at 72hours post fertilization. The transparency of the zebrafish embryo facilitates the studies in genetic. They can be easily manipulated genetically through the use of transgenic technology and gene knockdown via morpholinomodified antisense oligonucleotides mos. In vivo testing of micrornamediated gene knockdown in. Properties of gene knockdown system by vectorbased sirna. The expression of cartilagerelated genes in the tbx5a morphant was analyzed by dna microarray, immunostaining, and thinsection histology to examine the detailed distribution of the extracellular. Effective heritable gene knockdown in zebrafish using.
Crisprcas9mediated knockin and knockout in zebrafish. Jun 23, 2010 gene knockdown in embryos and the production of transgenic strains are now standard techniques being used to assess physiology. Gene knockdown is an experimental technique by which the expression of one or more of an organisms genes is reduced. A morpholino, also known as a morpholino oligomer and as a phosphorodiamidate morpholino oligomer pmo, is a type of oligomer molecule colloquially, an oligo used in molecular biology to modify gene expression. Current methods in zebrafish research gene tools, llc. However, methodology that allows spatiotemporally knockdown of gene expression in various model organisms such as zebrafish has not been well established, which largely limits the. Currently, the most common method to knockdown genes in zebrafish utilizes antisense morpholinos to block mrna translation, inhibit premrna splicing, or interfere with mirna function 32,35. Expanding the crispr toolbox in zebrafish for studying. Generating stable knockout zebrafish lines by deleting large.
For our knockdown experiments, we used zebrafish lines expressing egfp from. We show here that antisense, morpholinomodified oligonucleotides morpholinos are effective and specific translational inhibitors in. Pdf the sequencing of the zebrafish genome should be completed by the end of 2002. The development of mrna knockdown technologies for use in vertebrate organisms such as zebrafish has been limited. Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention.
We find that the zebrafish hoxb1 duplicates have equivalent functions to mouse hoxb1 and its paralogue hoxa1. Targeted gene knockdown in zebrafish using negatively charged. This book will be of keen interest not only to the typical readers of fish physiology but also to biomedical researchers, toxicologists and developmental biologists. While the general breeding and handling of zebrafish are detailed in the zebrafish book 9, this article highlights the important tools and methodology available in zebrafish research such as microinjection, morpholino knockdown, and transplantation, and discusses their applications. Pectoral fin anomalies in tbx5a knockdown zebrafish embryos. Use of sirnas against zebrafish lamin a, lamin b2 and the motor protein eg5, led to knockdown of the target genes with the specific phenotypes expected from prior studies in mammalian cells. Effective targeted gene knockdown in zebrafish nature. Background reduced expression of developmentally important genes and tumor suppressors due to haploinsufficiency or epigenetic suppression has been shown to contribute to the pathogenesis of various malignancies. This book will be of keen interest not only to the typical readers of. In contrast injection of lamin a, gl2 and egfp sirnas into zebrafish embryos resulted in morphological defects, abnormal development and early death of. Department of biomolecular medicine, ghent university, belgium. A ribozymemediated, gene knockdown strategy for the.
Functional knockdown of zebrafish tbx5a causes hypoplasia or aplasia of pectoral fins. Transient gene knockdown in zebrafish larvae is readily accomplished through the deployment of morpholino oligonucleotides mos bill et al. Crisprcasd induces efficient mrna knockdown in animal. Part of the research and perspectives in neurosciences book series. Despite being a popular and versatile model organism in which to study development and model disease, the use of zebrafish has been hampered by the lack of a reliable, stable and costeffective method of gene knockdown. The zebrafish genome contains a gene encoding an ortholog of the human elovl4, zfelovl4. We have used a morpholinobased knockdown approach to investigate the functions of a pair of zebrafish hox gene duplicates, hoxb1a and hoxb1b, which are expressed during development of the hindbrain. Currently, the most common method to knockdown genes in zebrafish utilizes antisense morpholinos to block mrna translation, inhibit premrna splicing, or interfere with mirna function32,35. Mos have been widely used in studies of zebrafish development, in part because of the lack until recently of methods for generating targeted null mutants as well as the accessibility of the mo technique to most laboratories. The sequencing of the zebrafish genome should be completed by the end of 2002. The structures of these 4 zebrafish igf genes and their transcripts have been determined. Nanoparticlemediated sirna genesilencing in adult zebrafish.
A database of zebrafish morpholinos is available at zfin, the zebrafish information network. Gene knockdown by morpholinomodified oligonucleotides in the. In summary, we have identified the conserved gucy2f ortholog gene in zebrafish, which is highly expressed in the retina, have shown that targeting this gene with antisense oligonucleotides leads to significantly decreased visual function in zebrafish larvae and leads to retinal photoreceptor changes. It is therefore not surprising that the discovery of rnai as an exploitable method of posttranscriptional gene regulation has created a lot of excitement within the zebrafish research community. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid. They further showed that knockdown of igf2a and 2b by antisense. Heritable and lineagespecific gene knockdown in zebrafish. The use of rnai technologies for gene knockdown in zebrafish.
Characterization of zebrafish dysferlin by morpholino knockdown. Gucy2f zebrafish knockdown a model for gucy2drelated. Love, journaljournal of biomedicine and biotechnology, year2012, volume2012. Zebrafish as a model to understand human genetic diseases. Targeted rna knockdown by crrna guided csm in zebrafish biorxiv. Here we report a protocol for performing nanoparticlemediated sirna gene silencing in adult zebrafish heart, thus providing a new lossoffunction method for studying adult organs in zebrafish and other model organisms.
Each target site had a fivestar ranking based on the ranking system using the invitrogen blockit rnai designer, which ensured that the designed mirnas would form the most effective rnai molecules. Here we investigate the knockdown efficiency of three synthetic mirnaexpressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. A guide for the laboratory use of zebrafish danio rerio. The other is that it is a novel igf as proposed by wang et. These can be reverted to wildtype gene expression with the appropriate splicemodifying morpholino oligo. Here, we report a convenient way of creating 3 zebrafish gene mutant lines with. The zebrafish system offers many unique opportunities for the study of molecular biology. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon older literature referred to zebrafish as brachydanio rerio.
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