The Experts below are selected from a list of 50037 Experts worldwide ranked by ideXlab platform
Vibha Srivastava - One of the best experts on this subject based on the ideXlab platform.
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Site-specific gene integration in rice genome mediated by the FLP-FRT Recombination System.
Plant biotechnology journal, 2010Co-Authors: Soumen Nandy, Vibha SrivastavaAbstract:Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific Recombination Systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT Recombination System has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT System for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT System for site-specific gene integration in plants using rice as a model.
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Utility of the FLP-FRT Recombination System for genetic manipulation of rice
Plant Cell Reports, 2005Co-Authors: Parthiban Radhakrishnan, Vibha SrivastavaAbstract:To develop an FLP- FRT Recombination System- (derived from 2 μ plasmid of Saccharomyces cerevisiae ) based marker gene removal application for rice, we introduced the gene for FLP recombinase, under the control of the maize ubiquitin-1 promoter, into the rice genome. FLP activity was monitored in callus and regenerated plants by an assay based on the deletion of the FRT -flanked DNA fragment, leading to the activation of the β-glucuronidase gene. FLP activity was detected both in the callus and leaves of some of the transgenic lines. Based on our comparison of the Recombination efficiency of the FLP- FRT System expressed in the transgenic lines with that of the widely used Cre- lox System (derived from bacteriophage P1), we suggest that the FLP- FRT System is a useful tool for the genetic manipulation of rice.
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Utility of the FLP-FRT Recombination System for genetic manipulation of rice.
Plant cell reports, 2004Co-Authors: Parthiban Radhakrishnan, Vibha SrivastavaAbstract:To develop an FLP-FRT Recombination System- (derived from 2 mu plasmid of Saccharomyces cerevisiae) based marker gene removal application for rice, we introduced the gene for FLP recombinase, under the control of the maize ubiquitin-1 promoter, into the rice genome. FLP activity was monitored in callus and regenerated plants by an assay based on the deletion of the FRT-flanked DNA fragment, leading to the activation of the beta-glucuronidase gene. FLP activity was detected both in the callus and leaves of some of the transgenic lines. Based on our comparison of the Recombination efficiency of the FLP-FRT System expressed in the transgenic lines with that of the widely used Cre-lox System (derived from bacteriophage P1), we suggest that the FLP-FRT System is a useful tool for the genetic manipulation of rice.
Ulrich Kück - One of the best experts on this subject based on the ideXlab platform.
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application of the saccharomyces cerevisiae flp frt Recombination System in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes
Applied and Environmental Microbiology, 2010Co-Authors: Katarina Kopke, Birgit Hoff, Ulrich KückAbstract:To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT Recombination System from the yeast Saccharomyces cerevisiae for marker recycling. We tested this System in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful Recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional Recombination System be generated in P. chrysogenum. As a proof of application of this System, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT System, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT Recombination System in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT Recombination System as a molecular tool for the genetic manipulation of filamentous fungi.
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Application of the Saccharomyces cerevisiae FLP/FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes
Applied and environmental microbiology, 2010Co-Authors: Katarina Kopke, Birgit Hoff, Ulrich KückAbstract:To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT Recombination System from the yeast Saccharomyces cerevisiae for marker recycling. We tested this System in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful Recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional Recombination System be generated in P. chrysogenum. As a proof of application of this System, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT System, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT Recombination System in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT Recombination System as a molecular tool for the genetic manipulation of filamentous fungi.
Hideo Takahashi - One of the best experts on this subject based on the ideXlab platform.
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Site-specific Recombination System based on actinophage TG1 integrase for gene integration into bacterial genomes
Applied microbiology and biotechnology, 2010Co-Authors: Nobutaka Hirano, Hideo Takahashi, Tetsurou Muroi, Yoshihiko Kihara, Ryuichi Kobayashi, Mitsuru HarukiAbstract:Phage integrases are enzymes that catalyze unidirectional site-specific Recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific Recombination System based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific Recombination System that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the Recombination efficiencies increased by a factor of ~10(1-3) in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This System thus may be beneficial to genome engineering for a wide variety of bacterial species.
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the site specific Recombination System of actinophage tg1
Fems Microbiology Letters, 2009Co-Authors: Kentaro Morita, Naoki Fusada, Mamoru Komatsu, Nobutaka Hirano, Tomoyuki Yamamoto, Haruo Ikeda, Hideo TakahashiAbstract:Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attPTG1) and bacterial (attBTG1) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attBTG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attBTG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (intTG1) was located adjacent to the attPTG1 site. TG1 integrase deduced from the intTG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific Recombination Systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.
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The site‐specific Recombination System of actinophage TG1
FEMS microbiology letters, 2009Co-Authors: Kentaro Morita, Naoki Fusada, Mamoru Komatsu, Nobutaka Hirano, Tomoyuki Yamamoto, Haruo Ikeda, Hideo TakahashiAbstract:Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attPTG1) and bacterial (attBTG1) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attBTG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attBTG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (intTG1) was located adjacent to the attPTG1 site. TG1 integrase deduced from the intTG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific Recombination Systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.
Katarina Kopke - One of the best experts on this subject based on the ideXlab platform.
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application of the saccharomyces cerevisiae flp frt Recombination System in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes
Applied and Environmental Microbiology, 2010Co-Authors: Katarina Kopke, Birgit Hoff, Ulrich KückAbstract:To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT Recombination System from the yeast Saccharomyces cerevisiae for marker recycling. We tested this System in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful Recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional Recombination System be generated in P. chrysogenum. As a proof of application of this System, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT System, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT Recombination System in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT Recombination System as a molecular tool for the genetic manipulation of filamentous fungi.
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Application of the Saccharomyces cerevisiae FLP/FRT Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes
Applied and environmental microbiology, 2010Co-Authors: Katarina Kopke, Birgit Hoff, Ulrich KückAbstract:To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/FRT Recombination System from the yeast Saccharomyces cerevisiae for marker recycling. We tested this System in the penicillin producer Penicillium chrysogenum using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the FRT sequences in direct repeat orientation (FRT-nat1 cassette) into a P. chrysogenum recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized P. chrysogenum flp (Pcflp) recombinase gene, were transferred into the P. chrysogenum strain carrying the FRT-nat1 cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful Recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional Recombination System be generated in P. chrysogenum. As a proof of application of this System, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/FRT System, we produced a flipper cassette carrying the FRT sites as well as the Pcflp gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/FRT Recombination System in other fungi was further demonstrated by marker recycling in the ascomycete Sordaria macrospora. Here, we discuss the application of the optimized FLP/FRT Recombination System as a molecular tool for the genetic manipulation of filamentous fungi.
Nobutaka Hirano - One of the best experts on this subject based on the ideXlab platform.
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Site-specific Recombination System based on actinophage TG1 integrase for gene integration into bacterial genomes
Applied microbiology and biotechnology, 2010Co-Authors: Nobutaka Hirano, Hideo Takahashi, Tetsurou Muroi, Yoshihiko Kihara, Ryuichi Kobayashi, Mitsuru HarukiAbstract:Phage integrases are enzymes that catalyze unidirectional site-specific Recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific Recombination System based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific Recombination System that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the Recombination efficiencies increased by a factor of ~10(1-3) in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This System thus may be beneficial to genome engineering for a wide variety of bacterial species.
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the site specific Recombination System of actinophage tg1
Fems Microbiology Letters, 2009Co-Authors: Kentaro Morita, Naoki Fusada, Mamoru Komatsu, Nobutaka Hirano, Tomoyuki Yamamoto, Haruo Ikeda, Hideo TakahashiAbstract:Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attPTG1) and bacterial (attBTG1) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attBTG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attBTG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (intTG1) was located adjacent to the attPTG1 site. TG1 integrase deduced from the intTG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific Recombination Systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.
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The site‐specific Recombination System of actinophage TG1
FEMS microbiology letters, 2009Co-Authors: Kentaro Morita, Naoki Fusada, Mamoru Komatsu, Nobutaka Hirano, Tomoyuki Yamamoto, Haruo Ikeda, Hideo TakahashiAbstract:Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attPTG1) and bacterial (attBTG1) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attBTG1 was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attBTG1 sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (intTG1) was located adjacent to the attPTG1 site. TG1 integrase deduced from the intTG1 gene was a protein of 619 amino acids having a high sequence similarity to φC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific Recombination Systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.
