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Narayana M. Upadhyaya - One of the best experts on this subject based on the ideXlab platform.
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Double-right-border (DRB) binary vectors for producing Selectable Marker-free transgenic rice.
2003Co-Authors: X. R. Zhou, J. Gorden, K. Ramm, X. R. Shen, Z. X. Gong, Narayana M. Upadhyaya, G. S. Khush, D. S. BrarAbstract:The continued presence of Selectable Marker genes, especially antibiotic and herbicide resistance genes, in transgenic plants to be released into the environment is of increasing public concern. Techniques such as the Cre/LoxP system of P1 bacteriophage, the FLP/FRT system, or the use of super binary vectors with two sets of T-DNA border sequences have been employed by researchers to produce Selectable Marker-free (SMF) transgenic progenies. We have further extended the twin T-DNA concept by using a double-right-border (DRB) binary vector. The structure of the DRB binary vector is as follows: RB1-Selectable Marker-RB2-GOI (gene of interest)-LB (left border). The assumption here is that two types of inserts (RB1 to LB and RB2 to LB) will be delivered and integrated into the genome and that, in the subsequent progenies, the two will segregate, thus allowing selection of SMF transgenic plants (RB2 to LB insert). We have tested this concept using two Selectable Marker genes (hph and bar) and have successfully segregated the second Selectable Marker gene from the first. Using the DRB binary vector system, we have recovered SMF transgenic lines containing a rice ragged stunt virus-derived synthetic resistance gene in two rice cultivars (Jarrah and Xu Shui) among segregating T 1 progeny plants. The SMF progenies of four out of eight Jarrah transgenic lines contained RRSVS5AS transgenes at a frequency of 50–100%. We are now using this system to stack two more useful genes in these rice cultivars.
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generation of Selectable Marker free transgenic rice using double right border drb binary vectors
Functional Plant Biology, 2001Co-Authors: X. R. Zhou, Z. X. Gong, Narayana M. UpadhyayaAbstract:Currently employed transformation systems require Selectable Marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these Selectable Markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce Selectable Marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a Selectable Marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the Selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two Selectable Marker genes and successfully obtained progeny plants in which the second Selectable Marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.
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Generation of Selectable Marker-free transgenic rice using double right-border (DRB) binary vectors
Functional Plant Biology, 2001Co-Authors: X. R. Zhou, Z. X. Gong, Narayana M. UpadhyayaAbstract:Currently employed transformation systems require Selectable Marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these Selectable Markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce Selectable Marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a Selectable Marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the Selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two Selectable Marker genes and successfully obtained progeny plants in which the second Selectable Marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny
Ralph Bock - One of the best experts on this subject based on the ideXlab platform.
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A bifunctional aminoglycoside acetyltransferase/phosphotransferase conferring tobramycin resistance provides an efficient Selectable Marker for plastid transformation
Plant Molecular Biology, 2017Co-Authors: Iman Tabatabaei, Ralph BockAbstract:Key message A new Selectable Marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA , and produces no background of spontaneous resistance mutants. Abstract More than 25 years after its development for Chlamydomonas and tobacco, the transformation of the chloroplast genome still represents a challenging technology that is available only in a handful of species. The vast majority of chloroplast transformation experiments conducted thus far have relied on a single Selectable Marker gene, the spectinomycin resistance gene aadA . Although a few alternative Markers have been reported, the aadA has remained unrivalled in efficiency and is, therefore, nearly exclusively used. The development of new Marker genes for plastid transformation is of crucial importance to all efforts towards extending the species range of the technology as well as to those applications in basic research, biotechnology and synthetic biology that involve the multistep engineering of plastid genomes. Here, we have tested a bifunctional resistance gene for its suitability as a Selectable Marker for chloroplast transformation. The bacterial enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia possesses an N-terminal acetyltransferase domain and a C-terminal phosphotransferase domain that can act synergistically and detoxify aminoglycoside antibiotics highly efficiently. We report that, in combination with selection for resistance to the aminoglycoside tobramycin, the aac(6 ′ )-Ie / aph(2 ″ )-Ia gene represents an efficient Marker for plastid transformation in that it produces similar numbers of transplastomic lines as the spectinomycin resistance gene aadA . Importantly, no spontaneous antibiotic resistance mutants appear under tobramycin selection.
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a bifunctional aminoglycoside acetyltransferase phosphotransferase conferring tobramycin resistance provides an efficient Selectable Marker for plastid transformation
Plant Molecular Biology, 2017Co-Authors: Iman Tabatabaei, Ralph BockAbstract:Key message A new Selectable Marker gene for stable transformation of the plastid genome was developed that is similarly efficient as the aadA, and produces no background of spontaneous resistance mutants.
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Chloramphenicol acetyltransferase as Selectable Marker for plastid transformation.
Plant molecular biology, 2010Co-Authors: Stephanie Ruf, Ralph BockAbstract:Chloroplast transformation remains a demanding technique and is still restricted to relatively few plant species. The limited availability of Selectable Marker genes and the lack of selection Markers that would be universally applicable to all plant species represent some of the most serious technical problems involved in extending the species range of plastid transformation. Here we report the development of the chloramphenicol acetyltransferase gene cat as a new Selectable Marker for plastid transformation. We show that, by selecting for chloramphenicol resistance, tobacco chloroplast transformants are readily obtained. Transplastomic lines quickly reach the homoplasmic state (typically in one additional regeneration round), accumulate the chloramphenicol acetyltransferase enzyme to high levels and transmit their plastid transgenes maternally into the next generation. No spontaneous antibiotic resistance mutants appear upon chloramphenicol selection. Several lines of evidence support the assumption that plant mitochondria are also sensitive to chloramphenicol suggesting that the chloramphenicol acetyltransferase may be a good candidate Selectable Marker for plant mitochondrial transformation.
X. R. Zhou - One of the best experts on this subject based on the ideXlab platform.
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Double-right-border (DRB) binary vectors for producing Selectable Marker-free transgenic rice.
2003Co-Authors: X. R. Zhou, J. Gorden, K. Ramm, X. R. Shen, Z. X. Gong, Narayana M. Upadhyaya, G. S. Khush, D. S. BrarAbstract:The continued presence of Selectable Marker genes, especially antibiotic and herbicide resistance genes, in transgenic plants to be released into the environment is of increasing public concern. Techniques such as the Cre/LoxP system of P1 bacteriophage, the FLP/FRT system, or the use of super binary vectors with two sets of T-DNA border sequences have been employed by researchers to produce Selectable Marker-free (SMF) transgenic progenies. We have further extended the twin T-DNA concept by using a double-right-border (DRB) binary vector. The structure of the DRB binary vector is as follows: RB1-Selectable Marker-RB2-GOI (gene of interest)-LB (left border). The assumption here is that two types of inserts (RB1 to LB and RB2 to LB) will be delivered and integrated into the genome and that, in the subsequent progenies, the two will segregate, thus allowing selection of SMF transgenic plants (RB2 to LB insert). We have tested this concept using two Selectable Marker genes (hph and bar) and have successfully segregated the second Selectable Marker gene from the first. Using the DRB binary vector system, we have recovered SMF transgenic lines containing a rice ragged stunt virus-derived synthetic resistance gene in two rice cultivars (Jarrah and Xu Shui) among segregating T 1 progeny plants. The SMF progenies of four out of eight Jarrah transgenic lines contained RRSVS5AS transgenes at a frequency of 50–100%. We are now using this system to stack two more useful genes in these rice cultivars.
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generation of Selectable Marker free transgenic rice using double right border drb binary vectors
Functional Plant Biology, 2001Co-Authors: X. R. Zhou, Z. X. Gong, Narayana M. UpadhyayaAbstract:Currently employed transformation systems require Selectable Marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these Selectable Markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce Selectable Marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a Selectable Marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the Selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two Selectable Marker genes and successfully obtained progeny plants in which the second Selectable Marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny <50% of plants contained the RRSV transgene. Thus, we have developed an efficient vector for producing SMF plants that allows straightforward cloning of any GOIs in comparison with the published ‘twin T-DNA’ vectors.
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Generation of Selectable Marker-free transgenic rice using double right-border (DRB) binary vectors
Functional Plant Biology, 2001Co-Authors: X. R. Zhou, Z. X. Gong, Narayana M. UpadhyayaAbstract:Currently employed transformation systems require Selectable Marker genes encoding antibiotic or herbicide resistance, along with the gene of interest (GOI), to select transformed cells from among a large population of untransformed cells. The continued presence of these Selectable Markers, especially in food crops such as rice (Oryza sativa L.), is of increasing public concern. Techniques based on DNA recombination and Agrobacterium-mediated co-transformation with two binary vectors in a single or two different Agrobacterium strains, or with super-binary vectors carrying two sets of T-DNA border sequences (twin T-DNA vectors), have been employed by researchers to produce Selectable Marker-free (SMF) transgenic progeny. We have developed a double right-border (DRB) binary vector carrying two copies of T-DNA right-border (RB) sequences flanking a Selectable Marker gene, followed by a GOI and one copy of the left border sequence. Two types of T-DNA inserts, one initiated from the first RB containing both the Selectable gene and the GOI, and the other from the second RB containing only the GOI, were expected to be produced and integrated into the genome. In the subsequent generation, these inserts could segregate away from each other, allowing the selection of the progeny with only the GOI. We tested this vector using two Selectable Marker genes and successfully obtained progeny plants in which the second Selectable Marker gene segregated away from the first. Using the DRB binary vector system, we recovered SMF transgenic lines containing a rice ragged stunt virus (RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu Shui. Approximately 36–64% of the primary transformants of these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny
Jingfu Li - One of the best experts on this subject based on the ideXlab platform.
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inducible excision of Selectable Marker gene from transgenic plants by the cre lox site specific recombination system
Transgenic Research, 2005Co-Authors: Yong Wang, Bojun Chen, Yuanlei Hu, Jingfu LiAbstract:In a plant transformation process, it is necessary to use Marker genes that allow the selection of regenerated transgenic plants. However, Selectable Marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a Marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and Selectable Marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a Selectable Marker gene, is easy to adopt and provides a promising approach to generate Marker-free transgenic plants.
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Inducible excision of Selectable Marker gene from transgenic plants by the cre/lox site-specific recombination system.
Transgenic Research, 2005Co-Authors: Yong Wang, Bojun Chen, Yuanlei Hu, Jingfu LiAbstract:In a plant transformation process, it is necessary to use Marker genes that allow the selection of regenerated transgenic plants. However, Selectable Marker genes are generally superfluous once an intact transgenic plant has been established. Furthermore, they may cause regulatory difficulties for approving transgenic crop release and commercialization. We constructed a binary expression vector with the Cre/lox system with a view to eliminating a Marker gene from transgenic plants conveniently. In the vector, recombinase gene cre under the control of heat shock promoter and Selectable Marker gene nptII under the control of CaMV35S promoter were placed between two lox P sites in direct orientation, while the gene of interest was inserted outside of the lox P sites. By using this vector, both cre and nptII genes were eliminated from most of the regenerated plants of primary transformed tobacco through heat shock treatment, while the gene of interest was retained and stably inherited. This autoexcision strategy, mediated by the Cre/lox system and subjected to heat shock treatment to eliminate a Selectable Marker gene, is easy to adopt and provides a promising approach to generate Marker-free transgenic plants.
William W. Metcalf - One of the best experts on this subject based on the ideXlab platform.
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The streptothricin acetyltransferase (sat) gene as a positive Selectable Marker for methanogenic archaea.
FEMS microbiology letters, 2019Co-Authors: Kristen R. Farley, William W. MetcalfAbstract:A repertoire of sophisticated genetic tools has significantly enhanced studies of Methanosarcina genera, yet the lack of multiple positive Selectable Markers has limited the types of genetic experiments that can be performed. In this study, we report the development of an additional positive selection system for Methanosarcina that utilizes the antibiotic nourseothricin and the Streptomyces rochei streptothricin acetyltransferase (sat) gene, which may be broadly applicable to other groups of methanogenic archaea. Nourseothricin was found to inhibit growth of four different methanogen species at concentrations ≤300 μg/ml in liquid or on solid media. Selection of nourseothricin resistant transformants was possible in two genetically tractable Methanosarcina species, M. acetivorans and M. barkeri, using the sat gene as a positive Selectable Marker. Additionally, the sat Marker was useful for constructing a gene deletion mutant strain of M. acetivorans, emphasizing its utility as a second positive Selectable Marker for genetic analyses of Methanosarcina genera. Interestingly, two human gut-associated methanogens Methanobrevibacter smithii and Methanomassillicoccus luminyensis were more sensitive to nourseothricin than either Methanosarcina species, suggesting the nourseothricin-sat gene pair may provide a robust positive selection system for development of genetic tools in these and other methanogens.
