The Experts below are selected from a list of 141 Experts worldwide ranked by ideXlab platform
Ulrich Technau - One of the best experts on this subject based on the ideXlab platform.
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Meganuclease assisted generation of stable transgenics in the sea anemone nematostella vectensis
Nature Protocols, 2017Co-Authors: Eduard Renfer, Ulrich TechnauAbstract:The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on Meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the Meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4-5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis-regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression.
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Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis
Nature Protocols, 2017Co-Authors: Eduard Renfer, Ulrich TechnauAbstract:The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on Meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the Meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4–5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis -regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression. The Technau lab provides their protocol for the generation of stably transgenic sea anemones. An expression vector is digested with the Meganuclease I-SceI and then microinjected into embryos, where I-SceI mediates stable integration into the genome.
Eduard Renfer - One of the best experts on this subject based on the ideXlab platform.
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Meganuclease assisted generation of stable transgenics in the sea anemone nematostella vectensis
Nature Protocols, 2017Co-Authors: Eduard Renfer, Ulrich TechnauAbstract:The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on Meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the Meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4-5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis-regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression.
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Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis
Nature Protocols, 2017Co-Authors: Eduard Renfer, Ulrich TechnauAbstract:The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on Meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the Meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4–5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis -regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression. The Technau lab provides their protocol for the generation of stably transgenic sea anemones. An expression vector is digested with the Meganuclease I-SceI and then microinjected into embryos, where I-SceI mediates stable integration into the genome.
Hong Wei - One of the best experts on this subject based on the ideXlab platform.
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The Meganuclease I-SceI containing nuclear localization signal (NLS-I-SceI) efficiently mediated mammalian germline transgenesis via embryo cytoplasmic microinjection.
PloS one, 2014Co-Authors: Yong Wang, Xiao-yang Zhou, Peng-ying Xiang, Lu-lu Wang, Huan Tang, Fei Xie, Hong WeiAbstract:The Meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the native I-SceI molecule was not capable of facilitating transgenesis in mammalian embryos via cytoplasmic microinjection as it did in fish eggs. In contrast, the I-SceI molecule containing mammalian nuclear localization signal (NLS-I-SceI) was shown to be capable of transferring DNA fragments from cytoplasm into nuclear in porcine embryos, and cytoplasmic microinjection with NLS-I-SceI mRNA and circular I-SceI recognition sequence-containing transgene plasmids resulted in transgene expression in both mouse and porcine embryos. Besides, transfer of the cytoplasmically microinjected mouse and porcine embryos into synchronized recipient females both efficiently resulted in transgenic founders with germline transmission competence. These results provided a novel method to facilitate mammalian transgenesis using I-SceI, and using the NLS-I-SceI molecule, a simple, efficient and species-neutral transgenesis technology based on embryo cytoplasmic microinjection with minimal bio-safety concerns can be established for mammalian species. As far as we know, this is the first report for transgenic mammals derived from I-SceI-mediated transgenesis via embryo cytoplasmic microinjection.
Pascale Daran-lapujade - One of the best experts on this subject based on the ideXlab platform.
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Efficient simultaneous excision of multiple selectable marker cassettes using I-SceI-induced double-strand DNA breaks in Saccharomyces cerevisiae.
FEMS yeast research, 2014Co-Authors: Daniel Solis-escalante, Niels G. A. Kuijpers, Jack T. Pronk, Jean-marc Daran, Franka H. Van Der Linden, Pascale Daran-lapujadeAbstract:Large strain construction programs and functional analysis studies are becoming commonplace in Saccharomyces cerevisiae and involve construction of strains that carry multiple selectable marker genes. Extensive strain engineering is, however, severely hampered by the limited number of recyclable marker genes and by the reduced genome stability that occurs upon repeated use of heterologous recombinase-based marker removal methods. The present study proposes an efficient method to recycle multiple markers in S. cerevisiae simultaneously, thereby circumventing shortcomings of existing techniques and substantially accelerating the process of selection-excision. This method relies on artificial generation of double-strand breaks around the selection marker cassette by the Meganuclease I-SceI and the subsequent repair of these breaks by the yeast homologous recombination machinery, guided by direct repeats. Simultaneous removal of up to three marker cassettes was achieved with high efficiencies (up to 56%), suggesting that I-SceI-based marker removal has the potential to co-excise an even larger number of markers. This locus- and marker-independent method can be used for both dominant and auxotrophy-complementing marker genes. Seven pDS plasmids carrying various selectable markers, which can be used for PCR-based generation of deletion cassettes suited for I-SceI marker recycling, are described and made available to the scientific community.
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One-step assembly and targeted integration of multigene constructs assisted by the I-SceI Meganuclease in Saccharomyces cerevisiae
FEMS yeast research, 2013Co-Authors: Niels G. A. Kuijpers, Soultana Chroumpi, Tim Vos, Daniel Solis-escalante, Lizanne Bosman, Jack T. Pronk, Jean-marc Daran, Pascale Daran-lapujadeAbstract:In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The Meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes.
Robert M Grainger - One of the best experts on this subject based on the ideXlab platform.
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High-throughput transgenesis in Xenopus using I-SceI Meganuclease
Nature Protocols, 2006Co-Authors: Hajime Ogino, William B Mcconnell, Robert M GraingerAbstract:In this report we describe an easy, highly efficient transgenesis method for Xenopus . The method is very simple; a commercially available Meganuclease, I-SceI , is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis ) or 20% (in Xenopus laevis ) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis .
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Highly efficient transgenesis in Xenopus tropicalis using I-SceI Meganuclease
Mechanisms of development, 2006Co-Authors: Hajime Ogino, William B Mcconnell, Robert M GraingerAbstract:Abstract In this study, we report a highly efficient transgenesis technique for Xenopus tropicalis based on a method described first for Medaka. This simple procedure entails co-injection of Meganuclease I-SceI and a transgene construct flanked by two I-SceI sites into fertilized eggs. Approximately 30% of injected embryos express transgenes in a promoter-dependent manner. About 1/3 of such embryos show incorporation of the transgene at the one-cell stage and the remainder are ‘half-transgenics’ suggesting incorporation at the two-cell stage. Transgenes from both classes of embryos are shown to be transmitted and expressed in offspring. The procedure also works efficiently in Xenopus laevis. Because the needle injection procedure does not significantly damage embryos, a high fraction develop normally and can, as well, be injected with a second reagent, for example an mRNA or antisense morpholino oligonucleotide, thus allowing one to perform several genetic manipulations on embryos at one time. This simple and efficient technique will be a powerful tool for high-throughput transgenesis assays in founder animals, and for facilitating genetic studies in the fast-breeding diploid frog, X. tropicalis.