The relative ease of their application and the flexibility of both of these systems ensured their wide distribution and popular use. The stable genomic integration and expression of a large transgene is a major hurdle in gene therapy. Several research groups have developed flexible transgenesis. Bac inserts int o the zebrafish and mouse genomes 33. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with singlecopy integrations. We have taken advantage of this transposition system to develop gene trap and enhancer trap methods kawakami et al. Several unique attributes make zebrafish a popular model to study vertebrate development and human diseases dooley and zon, 2000.
Oct 16, 2009 transposon mediated bac transgenesis in zebrafish and mice. Kwan km, fujimoto e, grabher c, mangum bd, hardy me, campbell ds, parant jm, yost hj, kanki jp, chien cb. Transgenesis in strongyloides and related parasitic. Bac transgenesis has been a powerful tool to study the function of the genome, and gene regulation by distal cis regulatory elements. Rapid bac selection for tol2mediated transgenesis in zebrafish. Bacterial artificial chromosomes bacs are among the most widely used tools for studies of gene regulation and function in model vertebrates. Especially, the germline transmission frequency with the tol2 transposon system is highest among currently available transgenesis methods in zebrafish. Zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. Pdf transposonmediated bac transgenesis in zebrafish. Meganuclease and transposon mediated transgenesis in. Bacterial artificial chromosomes bacs are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Transgenic zebrafish expressing fluorescent proteins in. A toolbox to study epidermal cell types in zebrafish. Here, we applied these techniques to zebrafish danio rerio in order to obtain insights into the cellular architecture of the axial motor column in vertebrates.
Evaluating the biological relevance of putative enhancers. In this new method, the minimal sequences of the tol2 transposon were used to deliver precisely single copies of a 70 kb bac transgene to the zebrafish and mouse genomes. The enhancertrap comprised of a basal promoter egfp gene flanked by 0. Such a wide distribution suggests that elements of this family require no or few. May 15, 2009 zebrafish is well suited for transgenic manipulation since it is relatively easy to collect large numbers of embryos from adult fish. For efficient transgenesis, the genomic dna piece in the bac construct needs to be flanked by tol2 transposon sites, and the constructs should.
Notably, transposase mediated transgenesis has been used in cells that are difficult to transfect including human haematopoietic stem cells 32, 33 and hescs 3436. May 28, 20 bacterial artificial chromosome bac transgenesis and geneenhancer trapping are effective approaches for identification of genetically defined neuronal populations in the central nervous system cns. Pb mediated transgenesis results in a single copy of a stably inherited and expressed transgene. Oct 10, 2000 the tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other fishes. In some cases, new bands or changes in band size may be detected from one. Breeding based remobilization of tol2 transposon in xenopus tropicalis breeding based remobilization of tol2 transposon in xenopus tropicalis. Rapid bac selection for tol2mediated transgenesis in. Oct 16, 2009 transposonmediated bac transgenesis in zebrafish and mice article pdf available in bmc genomics 101. Transposonmediated transgenesis, transgenic rescue, and. In this work, piggybac containing green fluorescent protein gfp was transferred into the pacific oyster crassostrea gigas by spermmediated gene transfer with or without electroporation. Transposon constructs and highly efficient transgenesis in zebrafish.
Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked dna to transposon mediated integration. In recent years, meganuclease and transposon mediated transgenesis protocols had a major impact on research employing transgenic animals, in particular, fish and lower vertebrates but also mice. We present an enhanced system, comprising a bac vector retrofitted with piggybac dna transposon elements and att l. Transposon mediated bac transgenesis in zebrafish and mice. Most potential enhancers ens still lack evidence of actual en activity. Kawakami k, takeda h, kawakami n, kobayashi m, matsuda n, mishina m. However, the efficiency of bac transgenesis is generally low, and gene transfer to bac vectors by recombination. Transgenesis and gene trap methods in zebrafish by using.
Transposonmediated bac transgenesis in human es cells pdf. Enhancer trapping and annotation in zebrafish mediated with. Pdf bacterial artificial chromosomes bacs are widely used in studies of vertebrate gene regulation and function because they often closely. Novel hypophysiotropic agrp2 neurons and pineal cells. Notably, transposase mediated transgenesis has been used in cells that are difficult to transfect including human haematopoietic stem cells 32,33 and hescs 3436. Transposonmediated bac transgenesis in zebrafish nature. A number of bacs can be prepared in parallel, and injection of the bacs containing cfpyfprfp or gal4 cassettes allows for immediate testing of whether a particular bac will yield the desired result. A tc1mariner superfamily transposon, minos, has excision and transposition activities in ciona. Several approaches have been developed for introducing transgenes into the zebrafish germline, from the injection of naked dna to transposon mediated. Conversion of a human sirpa carrying bac into a piggybac transposon. These advantages have promoted bacs to the forefront as transgenic tools and now bac transgenesis has been successfully applied to produce a variety of transgenic animals, such as mice, rats, zebrafish and flies, as well as for studies of gene function, molecular complementation of mutations, identification of distant regulatory elements and. A transposonmediated gene trap approach identifies. Then we constructed a transposonbased gene trap vector containing a splice acceptor. Toward this end, several efforts have been put to increase the efficiency of the transgenesis in zebrafish thermes et al.
Consequently, we were encouraged to examine whether bac transgenesis in hescs could be facilitated by transposition. The african killifish nothobranchius furzeri is the shortestlived vertebrate that can be bred in captivity. First, we established a highly efficient transgenesis method in which a plasmid dna containing the tol2 transposon vector and the transposase mrna synthesized in vitro were coinjected into onecell stage embryos. Rapid bac selection for tol2mediated transgenesis in zebra. Transgenesis is a popular approach to study the function of genes in zebrafish. Background bacterial artificial chromosomes bacs are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of bac. Transposonmediated bac transgenesis in human es cells article pdf available in nucleic acids research 4019. Lemberger bsc angestrebter akademischer grad master of science msc wien, 20 studienkennzahl lt. Tol2 transposonmediated transgenesis in the midas cichlid. Notably, transposasemediated transgenesis has been used in cells that are difficult to transfect including human haematopoietic stem cells 32, 33 and hescs 3436.
Bacterial artificial chromosome transgenesis through pronuclear injection of fertilized mouse oocytes. From among a plethora of various gene delivery methods, the researcher must choose the right one according to availability for a given species and the precise application the transgenic animal is intended for. Identification of a functional transposase of the tol2. While several transposons have been shown to work in zebrafish including sleeping beauty and acds, the most popular tool for transgenesis is the tol2 transposon. Currently, the most popular one is the tol2based transposon system. Suster ml1, kikuta h, urasaki a, asakawa k, kawakami k. Bac transgenesis has been used extensively in a broad range of applications in mice, from studies of gene regulation to creating animal models of human disease 1,2,3. Transposonmediated bac transgenesis in zebrafish and mice. Genomewide analysis of tol2 transposon reintegration in. This chapter focuses on transgenesis and gene trap methods in zebrafish by using the tol2 transposable element. The tol2 transposon system should thus be used to develop novel transgenesis and insertional mutagenesis methods in zebrafish and possibly in other fishes.
In this new method, the minimal sequences of the tol2 transposon were used to deliver precisely single copies of a approximately 70 kb bac transgene to the zebrafish and mouse genomes. Pdf transposonmediated bac transgenesis in zebrafish and mice. A plasmid set for efficient bacterial artificial chromosome. Transposonmediated bac transgenesis in zebrafish and mice core. Pdf transposonmediated bac transgenesis in human es cells. The initial steps required for developing a piggybac mediated bac transposition. Achievement of transposon mediated germline transgenesis in a basal chordate, ciona intestinalis, is discussed.
Construction of transgenic zebrafish is rather easy by a method using the tol2 transposon system. Although transposon mediated enhancer trapping et is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. We report here development of a novel gene trap method in zebrafish using the tol2 transposon system. We mapped the bac insertion sites in the genome by standard pcr methods and confirmed transposase mediated integrations. Minos enables the creation of stable transgenic lines, enhancer detection, and insertional mutagenesis. Due to distinct target site preferences, the number of target loci that can be mutagenized by transposon vectors could dramatically increase by combining different transposon systems in genomewide screens. Isolated bac dna was coinjected with tol2 transposase mrna to create first transient and, subsequently, stable transgenic zebrafish lines. Biomed central page 1 of 7 page number not for citation purposes bmc genomics methodology article open access transposon mediated bac transgenesis in zebrafish and mice. The zebrafish danio rerio is a useful model for genetic studies of vertebrate development. The tol1 element belongs to the hat hoboactivatortam3 transposable element family koga et al.
Furthermore, tol2 transposition has been used to integrate a 66 kb transgene into zebrafish and mouse genomes 39. Apr 01, 2016 transgenesis of large dna constructs is essential for gene function analysis. Transposon mediated bac transgenesis is a new tool for precise delivery of bacs in both zebrafish and mice. Bac transgenic lines expressing live reporters such as gfp can enhance the usefulness of zebrafish in developmental and evolutionary studies. New technology for scanning the zebrafish appb gene locus with enhancer traps using bacs was reported in an earlier study, and is illustrated in figure 1. However, even if a transposon inserts into a gene, it may not have a mutagenic effect. The medaka tol2 element is a d na type transposon that is active in a wide variety of vertebrates 35. We show that the modified piggybac pb transposon system can be used to introduce a 207 kb genomic dna fragment containing the ror. Transposonmediated bac transgenesis in human es cells. Here we discuss how transposons work in general, the application of transposon systems to zebrafish, and share a method for routine transgenesis using the tol2 transposon system kawakami and shima, 1999, koga et al. Functional analyses in vivo have typically relied on.
The nowcommon tol2 transposable element was originally isolated from medaka, and tol2 transposon mediated transgenesis, our method of choice, represented a significant improvement in the efficacy of transgenesis compared to previous approaches. Using tol2 mediated transgenesis, one can generate hundreds to thousands of transposon insertions in the zebrafish genome quickly and easily. Using bac transgenesis in zebrafish to identify regulatory. Recently, bac transgenesis in both mice and zebrafish was further facilitated by development of the transposon mediated method using the tol2 element. When making transgenic animals, the higher efficiency of the transgenesis is beneficial. Altmetric transposonmediated bac transgenesis in zebrafish. Rapid bac selection for tol2 mediated transgenesis in zebrafish. Introduction in drosophila, genomic deletions induced by transposon excision of p or minos element are one of the most commonly used methods to disrupt genes of interest 1,2.
Genomic deletion induced by tol2 transposon excision in zebrafish. Transposon tools and methods in zebrafish kawakami 2005. Transgenesis in zebrafish with the tol2 transposon system. Here, we introduce the developing zebrafish epidermis as an excellent in vivo model system for studying a living. Toward this end, several efforts have been put to increase the ef. Transposon mediated bac transgenesis in zebrafish and mice maximiliano l suster1, kenta sumiyama2,3 and koichi kawakami1,3. Transposonmediated bac transgenesis in zebrafish and mice article pdf available in bmc genomics 101. Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. Here we report a stepbystep protocol for efficient bac transgenesis in zebrafish using the medaka tol2 transposon. Transgenesis in strongyloides and related parasitic nematodes. Transposon mediated transgenesis in a marine invertebrate. Gal4 dna from bac clones chori211266o5, chori1073398l10, chori733l2 are shown.
Artificial chromosome transgenesis reveals longdistance negative regulation of rag1 in zebrafish. Germline transgenesis of zebrafish using the medaka tol1. Founder rates percentage of germline founders among total adult fish screened for constructs of different sizes 35250 kb indicate size independence of tol2 mediated bac transgenesis within the typical size range of zebrafish bac libraries. Jul 16, 2018 as an effective method of transgenesis, the plasmid of piggybac transposon containing gfp piggybac transposon system has been widely used in various organisms but not yet in mollusks. The injected dna was integrated in the genome, and one out of 20 injected fish 5% transmitted the integrated dna to the next generation stuart et al. Cellular dissection of the spinal cord motor column by bac. Although the use of transgenesis in zebrafish and medaka is widespread, its use in other teleosts. Most studies on epithelia and carcinomas use cell culture or organisms where highresolution live imaging is inaccessible without invasive techniques. Transposon mediated bac transgenesis in zebrafish and mice transposon mediated bac transgenesis in zebrafish and mice. Conclusion the tol2 transposon has a surprisingly large cargo capacity that can be harnessed for bac transgenesis. Evaluating the biological relevance of the myriad putative regulatory noncoding sequences in vertebrate genomes represents a huge challenge. Evaluating the biological relevance of putative enhancers using tol2 transposonmediated transgenesis in zebra. Transposon tools and methods in zebrafish kawakami.
Recently, tol2 transposase mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome bac dna constructs into the genome of zebrafish. We have established a simple, nonviral protocol in three impo. Here we discuss how transposons work in general, the application of transposon systems to zebrafish, and share a method for routine transgenesis using the tol2 transposon system koga et al. Epithelia provide a crucial protective barrier for our organs and are also the sites where the majority of carcinomas form.
The transposon vector inserted in the genome could be transmitted to the f1. It avoids potential multicopy concatameric integrations, allowing confirmation of the completeness of the bac from end to end, as well as mapping of the genomic integration site. Zebrafish danio rerio has been used as a model animal to study vertebrate development by genetic approaches. Tol2mediated transgenesis, gene trapping, enhancer. Frontiers piggybac transposonmediated transgenesis in the. Holger knaut, developmental genetics program and kimmel center for stem cell biology, skirball institute of biomolecular medicine, new york university langone medical center, 540 first avenue, skirball institute 4. Transposonmediated genome manipulations in vertebrates. Mar 20, 2017 zebrafish agrp2 mrna is expressed in the pineal gland 28,29,30 and was suggested, on the basis of morpholino mediated knockdown experiments, to regulate background pigment adaptation for. Notably, transposase mediated transgenesis has been used in cells that are difficult to transfect including human haematopoietic stem cells 32,33 and hescs. Tol2 element which encodes a gene for a fully functional transposase capable of catalyzing transposition in the zebrafish germ lineage. The sb100x mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus mediated transgenesis.
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