Microrna375 overexpression disrupts cardiac development of. The text provides significant updates for working vivarium professionals maintaining zebrafish colonies, veterinarians responsible for. Overexpression of dyrk1a, a down syndrome candidate gene. Overexpression of edn1 in 293t cells enhanced cell proliferation and cell migration in in vitro and xenotransplantation assays and was accompanied with upregulation of several cell cycleproliferation and migrationspecific genes.
Identification of dmrt2a downstream genes during zebrafish early. Here, we demonstrate a robust microinjection technique for performing mrna overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish. Despite its importance, in vivo systems for examining gene silencing at cellular resolution have been lacking in developing vertebrates. Ab we cloned and sequenced the zebrafish danio rerio connexin43 cx43. Most of the approaches taken to date to express foreign genes in zebrafish are based on injection of mrna for transient expression throughout the embryo. Here we apply the teton system to induce the expression of a zebrafish microrna, dremir722, in differentiated hl60. Human and zebrafish dyrk1a protein sequence possess 75.
Jul 01, 2009 epigenetic regulation of transcriptional silencing is essential for normal development. Transgenics transgenic zebrafish have essentially had exogenous genes added to their genome and as such can be used for many different experimental applications such as stable overexpression, dominantnegative mutation expression, and general analysis of gene regulation. This study aimed to evaluate the potentially beneficial effects of alginate oligosaccharide aos on the modulation of immunometabolic pathways in highfatdiet hfdinduced obese zebrafish and the underlying mechanism. The well characterized growth cycle and fully sequenced genome of d. Effect of mutated ids overexpression on ids enzyme activity. The potent combination of crisprcas9 and zebrafish as a model organism offers enormous potential for research into human diseases caused by point mutations. Overexpression of a zebrafish homologue of the drosophila. A guide for the laboratory use of zebrafish danio rerio. Accordingly, in our zebrafish dvl mutants, deficiency of posterior and trunk tissues could be obtained by substantially reducing maternal dvl dosage. Apr 17, 20 the virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed.
To investigate the role of ccnf dysfunction in neurodegenerative disease, we aimed to develop. Overexpression a novel zebra fish spermatogenesisassociated. 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. Cnbp ameliorates treacher collins syndrome craniofacial. Bach1b have been reported and bach1b is the main transcription factor, which heterodimerized with mafk to repress target gene expression, including heme oxygenase1a hmox1a and zymogen 1618. Transient disruption of the normal function of a gene during development can be achieved by microinjecting mrna, dna or short chemically stabilized antisense oligomers, called morpholinos mos, into early zebrafish embryos.
Transient disruption of the normal function of a gene during development can be achieved by microinjecting mrna, dna or short chemically stabilized antisense oligomers, called morpholinos. Targeted gene expression in the zebrafish prechordal plate. Biology, husbandry, diseases, and research applications is a comprehensive work that fulfills a critical need for a thorough compilation of information on this species. Zebrafish gene editing and gene modulation for life sciences.
Sep 28, 2016 in this study, we established baff overexpression juveniles and zebrafish, and detected the expression of nine immunerelated genes iglc1, iglc2, iglc3, igd, igm, igzt, rag1, tcrac and il4 and the protooncogene bcl2 in both juveniles and zebrafish spleen using realtime qpcr. Overexpression of the forebrainspecific homeobox gene. The rapid embryonic development and high fecundity of zebrafish contribute to the great advantages of this model for the study of developmental genetics. In the present study, we searched for novel target genes for zebrafish nrf2 to. Potential applications for zebrafish products include protein expression experiments. A molecular toolbox for genetic manipulation of zebrafish. Overexpression of fish or fly nkx6 is sufficient to generate supernumerary.
May 31, 2019 the effects of knockdown or overexpression of crbn on brain development are quite similar to the phenotypes induced by knockdown or overexpression of the transcription regulators six3 and lhx2b in zebrafish ando et al. N1aicd cy31, suggesting that hey1 may not respond to n1aicd overexpression in zebrafish. Hypoxia can impair reproduction of fishes through the disruption of sex steroids. Transcriptional silencing and reactivation in transgenic. Wish for ntl or pax2a expression showed that embryos injected with either. To investigate the role of ccnf dysfunction in neurodegenerative disease, we aimed to develop novel transgenic zebrafish models that. In zebrafish there are three six3related genes, six3a, six3b and six7 19, 20. Overexpression a novel zebra fish spermatogenesisassociated gene 17 spata17 induces apoptosis in gc1 cells november 2010 molecular biology reports 386. Oct 18, 2018 advances in optimisation of the gene editing technique crisprcas9 in zebrafish disease models offer a new level of accuracy and specificity previously out of reach for research into human genetic.
Coinjectionconfirmation at 24 and 48 hpf pictures analyses showed that overexpression of mir1423p repressed expression of meis1. Danio reio, commonly known as zebrafish, is an important model organism in biology research. Alginate oligosaccharide aos improves immunometabolic. Dec 15, 2010 europe pmc is an archive of life sciences journal literature. Building on a previously published bioinformatics study 23, homology searches of the zebrafish genome show that zebrafish have an hsenosf1. Suppression and overexpression of adenosylhomocysteine. Mutational analysis of dishevelled genes in zebrafish. Hsd2, cyp19a, and cyp19b, were found to be differentially upregulated at 24 and 48 hpf following zhif1odd overexpression a mutant zebrafish hif1.
The defects of gonads and germ cells in down syndrome suggest that overexpression of dyrk1a has potential effect on primordial germ cells pgcs development. Zebrafish models for the functional genomics of neurogenetic. Ghr gene expression as a consequence of gh resistance. More than 30 years ago, the small freshwater fish danio rerio attracted the attention of scientists who searched for an easytohandle but yet complex vertebrate to analyze conserved gene functions. An increase of ros along with the overexpression of redoxresponsive genes was detected. Common applications of these methodologies allow analysis of gainoffunction gene overexpression or dominant active, lossoffunction gene knock down or dominant negative, mosaic analysis, lineagerestricted studies and cell tracing experiments. Zebrafish overexpression 1 developmental biology interactive. M was microinjected into the hindbrain ventricles of zebrafish larvae at 4 dpf and the larvae were double immunostained with antiacetylated. Here, we investigate the functions of olfm1 in zebrafish eye development. Differential gene expression analysis was assessed by deseq package. Red and green text donate genes with increased and decreased expression, respectively, in tgkrt4. Myf5 is a myogenic regulatory factor that functions in myogenesis. In order to analyse the function of this novel zinc finger gene during vertebrate development, we decided to identify a potential orthologue in the teleost fish danio.
Unlike the ubiquitously present ahcy genes, ahcyl1 genes were only. Its molecular structure contains dna bases attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. The zebrafish reference genome sequence and its relationship. Pdf overexpression of the forebrainspecific homeobox gene. Here we cloned two een genes designated as eena and eenb in zebrafish, which are assigned to linkage groups 8 and 2, respectively. The mllaf9 fusion gene, resulting from chromosomal translocation, often leads to acute myeloid leukemia with poor prognosis.
Can insectbased diets affect zebrafish danio rerio. For the overexpression of three zebrafish mutated ids mrnas corresponding to three known human mutated ids effects on the embryonic development of zebrafish in this study, we observed three major defective phenotypes. Igfi gene regulation in different genotypes of ghtransgenic zebrafish. Full text a molecular toolbox for genetic manipulation of zebrafish. Overexpression of fulllength olfm1, and especially its bmy form lacking the olfactomedin domain, increased the thickness of the optic nerve and produced a more extended projection field in the optic tectum compared with control embryos.
Bmp2b overexpression causes a pancreatictoliver fate switchaf dorsal views of hhex ac and pdx1 df expression at 44 hpf, comparing control a and d and bmp2boverexpressing embryos heat shock applied at the 8 b and e or 14 c and f somite stage. Functional characterization of the bcl2 gene family in. However, mrna injection risks expressing genes ectopically, such as in the case of synaptic genes, and is usually only efficient at embryonic stages, prior to mrna. Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. Transgenic zebrafish an overview sciencedirect topics. This simple approach is valid for ubiquitously and early expressed genes such as ap1s1, sod1, tardbp and members of the ubiquination and betasecretase complexes discussed above. As dmrt2a has a paralog gene, dmrt2b, with similar functions and expression pattern, we evaluated the possibility of redundancy. Targeted gene expression is a powerful tool for understanding gene function in vivo. Conservation of the nrf2mediated gene regulation of proteasome. Identification of 315 genes essential for early zebrafish. Microrna29c affects zebrafish cardiac development via. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling. In zebrafish, overexpression of gene products is typically accomplished ubiquitously, without temporal and spatial specificity.
Overexpression of edn1 in 293t cells enhanced cell proliferation and cell migration in in vitro and xenotransplantation assays and was accompanied with upregulation of several cell cycleproliferation and. Our data suggest that there are roughly 1,400 embryonicessential genes in the fish. The zebrafish ab line was used, and embryos and adult fish were raised according to the standard protocol described in the zebrafish book. Using oligonucleotide microarrays, gene expression signatures of zebrafish liver tumors were compared to normal zebrafish liver and human. Overexpression of mutant superoxide dismutase 1 causes a. Mutational analysis of dishevelled genes in zebrafish reveals. Thus, we overexpressed mir375 in a zebrafish model to clarify its effect on. Mutations in this gene have recently been linked to the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia.
The predicted protein sequence shows a high degree of sequence conservation. Oct 05, 2017 programmed cell death pcd is a conserved cellular process, which is essential during embryonic development, morphogenesis and tissue homeostasis. Dyrk1a, located on chromosome 21, is a major candidate gene of down syndrome ds, trisomy21, and its overexpression is associated with abnormal phenotype of down syndrome patients. Human mllaf9 overexpression induces aberrant hematopoietic. Cereblon control of zebrafish brain size by regulation of. Several zebrafish bcl2 genes have been previously reported in the literature. Jun 05, 20 to investigate whether transgenic overexpression of cdx1b, the functional equivalent of mammalian cdx2 in zebrafish, may lead to im of zebrafish esophageal squamous epithelium, a transgenic zebrafish system was developed by expressing cdx1b gene under the control of zebrafish keratin 5 promoter krt5p. Genetic engineering of zebrafish in cancer research. Mir1423p regulates cardiovascular system during zebrafish. Alginate oligosaccharide aos improves immunometabolic systems by inhibiting stoml2 overexpression in highfatdietinduced obese zebrafish v. We generated transgenic zebrafish lines expressing human tdp43, either the.
The sp7 gene encodes a zinc fingercontaining transcriptional factor expressed in osteoblasts so that it can be a marker of osteoblasts. Effect of mutated ids overexpression on ids enzyme. Developmental regulation and expression of the zebrafish. Morpholino gene knockdowns of genesis volume 30, issue 3 pages 89200 july 2001. Mllaf9 mrna was injected into zebrafish at the 12cell stage around 5ng per embryo. Functional characterization of the bcl2 gene family in the. The 11q23 of the mixed lineage leukemia 1 mll1 gene plays a crucial role in early embryonic development and hematopoiesis. Finally, a positive correlation between the expression of cnbp and tcof1 in mesenchymal cells from both control and tcs subjects was found. Conversely, human and zebrafish ahcyl1a mrna injection into. Thus, microinjection has been widely used for generating transgenic fish 1, 2, 3, analyzing gene function by overexpression of dna or rna 4, 5, 6 and mapping cell fate in early blastula embryos 7, 8. Wholemount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Overexpression of six3 in zebrafish embryos induced enlargement of the rostral forebrain, enhanced expression of pax2 in the optic stalk and led to a general disorganization of the brain. Here we show that ectopic expression of nkx6 genes in zebrafish or flies results in embryos.
Molecular cloning and expression analysis of a zebrafish novel zinc. The microarray data of triplicated assay were submitted to ncbi gene expression omnibus under accession numbers gsm542371 to. Transgenic overexpression of baff regulates the expression. The overexpression of dominantnegative brg1 blocks cardiac regeneration and leads to evident scar formation in zebrafish at 30 dpa. Combinatorial gene regulation by bmp and wnt in zebrafish. I have been trying to clone an about 6kb large gene in to a vector that later is gonna be used for mrna expression zebrafish. Here, we generated a zebrafish model expressing the human mllaf9 fusion gene.
In zebrafish, the wnt and bmp pathways interact to regulate the formation of the posterior body. Here, using zebrafish danio rerio embryos, we investigated i whether hypoxia can directly affect steroidogenesis independent of pituitary regulation via modulation of steroidogenic gene expression, and ii the role of leptin in hypoxiainduced disruption of steroidogenesis. Embryos were obtained by natural spawning and were maintained in e3 zebrafish water at 28. Overexpression of akt1 enhances adipogenesis and leads to. May 18, 2012 network of the deregulated genes in obsese transformed zebrafish. Injections are most frequently used for transient overexpression of proteins in. Sep 26, 2011 zebrafish also have a sequenced genome, allowing for easy gene finding and straightforward comparison of zebrafish genes to human genes 3033. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed. We describe a transgenic approach that allows monitoring of an epigenetically regulated fluorescent reporter in developing zebrafish and their progeny. For instance, zebrafish with the mutated myo18b gene can serve as a model for. An intronic microrna, mirin300, located within zebrafish myf5 intron i, has been reported to silence myf5 through the targeting of dickkopf3related gene dkk3r.
The conservation of protein sequence and developmental gene regulation would suggest that cx43. How to demonstrate protein overexpression in zebrafish. Figure 2 genomic organization and alternative splicing of ahcyl1 in zebrafish a and b and humans c and d. The e3 ubiquitin ligase protein, cyclin f encoded by ccnf has a role in substrate recognition for protein degradation within the ubiquitin proteasomal system. Ijms free fulltext overexpression of notch signaling. Liverspecific edn1 expression caused steatosis, fibrosis, glycogen accumulation, bile duct dilation, hyperplasia, and hcc in zebrafish. Amiodarone induces overexpression of similar to versican b. A, schematic representation of the zahcyl1 gene and the alternative 5. May 30, 2018 mllaf9 overexpression in zebrafish embryos and activation of mll downstream genes. Keywords hif1, hypoxia, steroidogenic enzyme genes, zebrafish, steroid.
Novel recessive mutations in msto1 cause cerebellar atrophy with. Fish were maintained as described in the zebrafish book. Overexpression of zebrafish ids mrna in fertilized eggs increases the enzymatic activity of ids in zebrafish embryos zebrafish embryos served as an in vivo platform to evaluate whether mutated ids contributes to mps ii and if so, to what degree. Enhancer trapping is more directed to the identification of novel regulative elements such as. The een extra eleven nineteen gene is one of the fusion partners of mixedlineage leukemia, located on chromosome 19p. Expression of sod1 wt, sod1 g93a, sod1 g37r and sod1 a4v in zebrafish embryos 30 hpf. The combined loss of zebrafish six3b and six7 function resulted in anophthalmia or microphthalmia 21.
Microinjection into zebrafish embryos springerlink. A microarray analysis of zebrafish embryos that overexpressed nrf2 revealed that. Transient overexpression during early zebrafish development up to 3 days is achieved by introducing the mrna encoding the protein of interest into the embryos during the first two hours of development. Manipulation of gene expression during zebrafish embryonic. Transgenic overexpression of cdx1b induces metaplastic. Overexpression baff regulates immune genes expression in zebrafish. To stabilize the injected mrna transcript, the regulatory mrna regions, such as 3 untranslated regions utr are used figure 1. Aug 06, 2018 in overexpression experiments, high levels of xdsh dvl2 activate posterior neural markers, whereas low levels induce the expression of anterior neural genes.
Epigenetic regulation of organ regeneration in zebrafish. Frontiers zebrafish models of neurodevelopmental disorders. Adenosylhomocysteine hydrolaselike protein 1 ahcyl1 is a novel intracellular. On the other hand, treacle depletion led to a lowering in the abundance of cnbp, a protein required for proper craniofacial development. Constitutive overexpression of the alslinked gene ccnf. Overexpression and knockdown of hypoxiainducible factor 1. Overexpression a novel zebra fish spermatogenesisassociated gene 17 spata17 induces apoptosis in gc1 cells. However, the yeast gal4uas system can be used for targeted gene expression in zebrafish. 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.
Transgenic overexpression of baff regulates the expression of. Overexpression of endothelin 1 triggers hepatocarcinogenesis. Disruption of either the six domain or the homeodomain abolish these effects, implying that these domains are essential for six3 gene function. May 07, 2009 microinjection is a wellestablished and effective method for introducing foreign substances into fertilized zebrafish embryos. Oct 09, 2020 results showed that while fish were perfectly able to cope with a 25% insect meal dietary inclusion, a 50% inclusion level caused the overexpression of genes involved in lipid metabolism, a general reduction in the number of spawned eggs, and differences in the frequency rate of previtellogenic oocytes, class iii, iv, oocytes and postovulatory. Transcriptomic analysis of zebrafish tdp43 transgenic. The cnbp overexpression rescued the tcs phenotype in a dosedependent manner by a roscytoprotective action that prevented the redoxresponsive genes upregulation but did not normalize the synthesis of rrnas. Geneedited zebrafish models take disease research to the. Overexpression of this gene following mrna injections leads to a reduction in the number of islet1 positive cells, which are assumed to be primary neurons, and. The gene consists of 18 exons e1e18, indicated by boxes.
The two variants, zahcyl1a and b, differ at the n terminus of the protein due to the alternative use of either exon 1 or exon 2. Here we describe 315 mutants and the corresponding genes. Aug 31, 2004 we completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development. Zebrafish offers important advantages as a genetic model organism such as a. Sep 29, 2000 in zebrafish, the expression patterns of a number of genes, including notch5, deltac, deltad, and notch6, clearly demonstrate that anteroposterior subdivisions are present in presumptive and formed somites fig. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Pcd participates in the elimination of unwanted or potentially harmful cells, and contributes in this way to the precise shaping of the developing embryo. Ectopic wnt signal determines the eyeless phenotype of. Expression of a novel growth hormone gene results in growth enhancement in transgeni. The effect of gh overexpression on ghr and igfi gene regulation. Injected embryos and control embryos were harvested at 48hpf. Feb 19, 2021 transient overexpression during early zebrafish development up to 3 days is achieved by introducing the mrna encoding the protein of interest into the embryos during the first two hours of development. Overexpression and knockdown of hypoxiainducible factor 1 disrupt the expression of steroidogenic enzyme genes and early embryonic development in zebrafish tianfeng tan1,2,3, richard man kit yu4, rudolf shiu sun wu1,5 and richard yuen chong kong1,3 1state key laboratory in marine pollution, city university of hong kong, hong kong sar. Transient knockdown and overexpression reveal a developmental.
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