The biosafety of genetically engineered plants has been of concernment in society and science in recent years. The issue of 35S promoter of CaMV has been contentious because of its wide use in plant genetic engineerin...The biosafety of genetically engineered plants has been of concernment in society and science in recent years. The issue of 35S promoter of CaMV has been contentious because of its wide use in plant genetic engineering. The debate on the safety and potential risks of the 35S promoter will be discussed here. Some of concerns are expressed about the dissemination of antibiotic_resistance genes and vector backbone sequences. Various methods and strategies are currently being developed for the marker gene excision and elimination of vector backbone sequences from transgenic plants. In this review, the CRE/ lox system which could get rid of the marker geens and vector backbone sequences will be discussed in detail. Advances in the research of the safety assessment of genetically modified plants using the CRE/ lox system will also be described.展开更多
Marker free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the selectable marker genes conferring antibiotic or herbicide resistance and so on. Furthermore,...Marker free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the selectable marker genes conferring antibiotic or herbicide resistance and so on. Furthermore, marker_free transgenic plants (MFTPs) have a number of special advantages, such as decreasing the concerns about safety of selectable marker and stacking transgenes progressively into transgenic plants, which significantly owns potential application value. Major approaches developed recently for obtaining MFTPs were reviewed in this paper.展开更多
The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different tim...The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site- specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.展开更多
This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breedin...This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper （Capsicum annuum L.）. Chili pepper inbred lines （A, D, E, and I） were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR[MB（MS mineral＋vitamine B5）＋BA（6-Benzyladenine） 5.0 mg·L^-1 ＋IAA（indoleacetic acid） 1.0 mg·L^-1＋GA3（gibberellic acid） 1.0mg·L^-1＋sucrose 3%＋agar 6.5g·L^-1] for 2d. The explants were infected by Agrobacterium tumefaciens when their OD600（optical density at 600 nm）reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB＋BA5.0 mg·L^-1＋IAA 1.0 mg·L^-1 ＋GA3 1.0 mg·L^-1＋sucrose 3% ＋agar 6.5 g·L^-1＋AS （acetosyringone） 200μmol·L^-1, these cotyledons with petiole were cultured on selective differentiation medium in the media MT[MB medium supplemented with BA [5.0 mg·L^-1＋ IAA 1.0 mg·L^-1＋ GA3 1.0 mg·L^-1＋ AgNO3 5.0 mg·L^-1＋ CW （coconut water） 5% ＋ Km （kanamycin） 65 mg·L^-1＋ Cb （carbenicillin） 500 mg·L^-1＋ 3% sucrose ＋ agar 6.5 g·L^-1].The Kmr （kanamycin resistant） bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar resul展开更多
Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanke...Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.展开更多
Chondroitin sulfate proteoglycan-4（CSPG4） is a surface component of two key cell types（oligodendrocyte progenitor cells（OPCs） and myeloid cells） present in lysolecithin-induced lesions in mouse spinal cord.Two t...Chondroitin sulfate proteoglycan-4（CSPG4） is a surface component of two key cell types（oligodendrocyte progenitor cells（OPCs） and myeloid cells） present in lysolecithin-induced lesions in mouse spinal cord.Two types of CSPG4 manipulations have been used to study the roles of these cells in myelin damage and repair：（1） OPC and myeloid-specific ablation of CSPG4,and（2） transplantation of enhanced green fluorescent protein（EGFP）-labeled progenitors to distinguish between bone marrow-derived macrophages and resident microglia.Ablation of CSPG4 in OPCs does not affect myelin damage,but decreases myelin repair,due to reduced proliferation of CSPG4-null OPCs that diminishes generation of mature oligodendrocytes for remyelination.Ablation of CSPG4 in myeloid cells greatly decreases recruitment of macrophages to spinal cord lesions,resulting in smaller initial lesions,but also in significantly diminished myelin repair.In the absence of macrophage recruitment,OPC proliferation is greatly impaired,again leading to decreased generation of myelinating oligodendrocytes.Macrophages may promote OPC proliferation via phagocytosis of myelin debris and/or secretion of factors that stimulate OPC mitosis.Microglia are not able to substitute for macrophages in promoting OPC proliferation.An additional feature of lesions in myeloid-specific CSPG4 null mice is the persistence of poorly-differentiated platelet-derived growth factor receptor α（PDGFRα） ＋ macrophages that may prolong damage.展开更多
文摘The biosafety of genetically engineered plants has been of concernment in society and science in recent years. The issue of 35S promoter of CaMV has been contentious because of its wide use in plant genetic engineering. The debate on the safety and potential risks of the 35S promoter will be discussed here. Some of concerns are expressed about the dissemination of antibiotic_resistance genes and vector backbone sequences. Various methods and strategies are currently being developed for the marker gene excision and elimination of vector backbone sequences from transgenic plants. In this review, the CRE/ lox system which could get rid of the marker geens and vector backbone sequences will be discussed in detail. Advances in the research of the safety assessment of genetically modified plants using the CRE/ lox system will also be described.
文摘Marker free is a rapidly developed strategy that offers a new approach for the elimination of public concerns caused by the selectable marker genes conferring antibiotic or herbicide resistance and so on. Furthermore, marker_free transgenic plants (MFTPs) have a number of special advantages, such as decreasing the concerns about safety of selectable marker and stacking transgenes progressively into transgenic plants, which significantly owns potential application value. Major approaches developed recently for obtaining MFTPs were reviewed in this paper.
文摘The current method for combining transgenes into a genome is through the assortment of independent loci, a classical operating system compatible with transgenic traits created by different developers, at different times and/or through different transformation techniques. However, as the number of transgenic loci increases over time, increasingly larger populations are needed to find the rare individual with the desired assortment of transgenic loci along with the non-transgenic elite traits. Introducing a transgene directly into a field cultivar would bypass the need to introgress the engineered trait. However, this necessitates separate transformations into numerous field cultivars, along with the characterization and regulatory approval of each independent transformation event. Reducing the number of segregating transgenic loci could be achieved if multiple traits are introduced at the same time, a preferred option if each of the many traits is new or requires re-engineering. If reengineering of previously introduced traits is not needed, then appending a new trait to an existing locus would be a rational strategy. The insertion of new DNA at a known locus can be accomplished by site- specific integration, through a host-dependent homology-based process, or a heterologous site-specific recombination system. Here, we discuss gene stacking through the use of site-specific recombinases.
基金supported by the National Key Technology R&D Program of China(2006BAD01A7-04-09)Guangdong Key Technology Program(2006B20201028),China
文摘This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/lox system, for heterosis breeding of chili pepper （Capsicum annuum L.）. Chili pepper inbred lines （A, D, E, and I） were transformed with Cre gene and Barnase gene situated between loxp, separately, by means of Agrobacterium co-culture. In this study, we had established a high transformation system by extensive study of affecting factors including genotype, selection of marker, and lethal dose. Cotyledon with petiole from 9-11-day-old seeding was pre-cultured on media MR[MB（MS mineral＋vitamine B5）＋BA（6-Benzyladenine） 5.0 mg·L^-1 ＋IAA（indoleacetic acid） 1.0 mg·L^-1＋GA3（gibberellic acid） 1.0mg·L^-1＋sucrose 3%＋agar 6.5g·L^-1] for 2d. The explants were infected by Agrobacterium tumefaciens when their OD600（optical density at 600 nm）reached 0.6-0.9. After co-cultured for 4-5 d on media MC [MB＋BA5.0 mg·L^-1＋IAA 1.0 mg·L^-1 ＋GA3 1.0 mg·L^-1＋sucrose 3% ＋agar 6.5 g·L^-1＋AS （acetosyringone） 200μmol·L^-1, these cotyledons with petiole were cultured on selective differentiation medium in the media MT[MB medium supplemented with BA [5.0 mg·L^-1＋ IAA 1.0 mg·L^-1＋ GA3 1.0 mg·L^-1＋ AgNO3 5.0 mg·L^-1＋ CW （coconut water） 5% ＋ Km （kanamycin） 65 mg·L^-1＋ Cb （carbenicillin） 500 mg·L^-1＋ 3% sucrose ＋ agar 6.5 g·L^-1].The Kmr （kanamycin resistant） bud rosettes were elongated on selective elongation medium and rooted on rooting medium. PCR and Southern blotting analysis of Kmr plantlet indicated that the foreign genes had been integrated into the genome of pepper. The transgenic plants with Cre gene developed well, blossomed out, and set fruit normally. The transgenic plants with Barnase gene grew well with normal appearance of flower, but they showed different fertility from complete sterility, partial sterility to complete fertility, and similar resul
基金the National Natural Science Foundation of China (30200185)the Science Foundation of Committee of Education of Chongqing Municipality,China (030208)
文摘Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.
基金supported by National Institutes of Health R01 CA095287(WBS)Sanford Burnham Prebys Medical Discovery Institute Lab Funding Initiative(WBS)
文摘Chondroitin sulfate proteoglycan-4（CSPG4） is a surface component of two key cell types（oligodendrocyte progenitor cells（OPCs） and myeloid cells） present in lysolecithin-induced lesions in mouse spinal cord.Two types of CSPG4 manipulations have been used to study the roles of these cells in myelin damage and repair：（1） OPC and myeloid-specific ablation of CSPG4,and（2） transplantation of enhanced green fluorescent protein（EGFP）-labeled progenitors to distinguish between bone marrow-derived macrophages and resident microglia.Ablation of CSPG4 in OPCs does not affect myelin damage,but decreases myelin repair,due to reduced proliferation of CSPG4-null OPCs that diminishes generation of mature oligodendrocytes for remyelination.Ablation of CSPG4 in myeloid cells greatly decreases recruitment of macrophages to spinal cord lesions,resulting in smaller initial lesions,but also in significantly diminished myelin repair.In the absence of macrophage recruitment,OPC proliferation is greatly impaired,again leading to decreased generation of myelinating oligodendrocytes.Macrophages may promote OPC proliferation via phagocytosis of myelin debris and/or secretion of factors that stimulate OPC mitosis.Microglia are not able to substitute for macrophages in promoting OPC proliferation.An additional feature of lesions in myeloid-specific CSPG4 null mice is the persistence of poorly-differentiated platelet-derived growth factor receptor α（PDGFRα） ＋ macrophages that may prolong damage.
