Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics

Selectable marker gene systems are vital for the development of transgenic crops. Since the creation of the first transgenic plants in the early 1980s and their subsequent commercialization worldwide over almost an entire decade, antibiotic and herbicide resistance selectable marker gene systems have been an integral feature of plant genetic modification. Without them, creating transgenic crops is not feasible on purely economic and practical terms. These systems allow the relatively straightforward identification and selection of plants that have stably incorporated not only the marker genes but also genes of interest, for example herbicide tolerance and pest resistance. Bacterial antibiotic resistance genes are also crucial in molecular biology manipulations in the laboratory. An unprecedented debate has accompanied the development and commercialization of transgenic crops. Divergent policies and their implementation in the European Union on one hand and the rest of the world on the other (industrialized and developing countries alike), have resulted in disputes with serious consequences on agricultural policy, world trade and food security. A lot of research effort has been directed towards the development of marker-free transformation or systems to remove selectable markers. Such research has been in a large part motivated by perceived problems with antibiotic resistance selectable markers; however, it is not justified from a safety point of view. The aim of this review is to discuss in some detail the currently available scientific evidence that overwhelmingly argues for the safety of these marker gene systems. Our conclusion, supported by numerous studies, most of which are commissioned by some of the very parties that have taken a position against the use of antibiotic selectable marker gene systems, is that there is no scientific basis to argue against the use and presence of selectable marker genes as a class in transgenic plants.     

Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments.

Plasmid pKC7, a derivative of pBR322, specifies resistance to both ampicillin and kanamycin. The DNA of this small plasmid (5.8 kb) contains unique sites for insertion of DNA cleaved with ten different restriction endonucleases. A detailed restriction endonuclease cleavage map is presented. The utility of this plasmid for cloning is discussed.     

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T₁ soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.     

Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker

We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.     

Genetically engineered resistance to organophosphate herbicides provides a new scoreable and selectable marker system for transgenic plants

Organophosphate hydrolase (OPH, E.C. 3.1.8.1; encoded by the bacterial opd gene) provides a new scoreable and selectable genetic marker system for use in plant cell culture and regenerated plant tissue. OPH hydrolyzes a wide range of substrates that produce visually detectable products, which can be readily quantified in biological tissues. A variety of different OP compounds, both herbicides and pesticides, have been identified as acceptable enzymatic substrates, which can be used to generate transgenic markers for various types of plant tissues. For example, transgenic leaf tissue was easily differentiated from non-transgenic tissue by a simple fluorescent assay utilizing the OP insecticide coroxon. Transformed callus and intact whole seed could be easily distinguished from non-transformed tissue using novel non-destructive methods which allowed callus or seeds to grow and/or to germinate after phenotypic scoring with non-herbicidal OP insecticides such as paraoxon. In addition to being used as a scoreable phenotypic markers with various OP pesticides, the OP compounds Haloxon and Bensulide (Bensumec-4LF) were effective as positive selection agents for callus and germinating seeds.     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999     

微生物遗传转化筛选标记及其生物安全性的研究进展

在分子生物技术中,筛选标记基因是遗传转化载体所必备的基本元件之一,其主要功能是在基因操作中进行目标克隆的筛选,以及在应用过程中通过选择压力维持基因重组性状。抗药基因是微生物遗传转化中常用的筛选标记,大肠杆菌载体和一般穿梭载体中通常带有抗药基因。带有抗药基因的工程菌可以被广泛地应用于酶和有机化学品的发酵生产,因为工业发酵过程是在封闭系统中进行的,并且最终产品需要经过提炼。但是当人们需要用基因改良的菌株进行食品和饲料加工、环境修复、病虫害生物防治时,抗药基因类筛选标记应该被禁止使用。因此,发展生物安全性筛选标记成为遗传转化技术推广应用中的一个技术关键。本文介绍常用作筛选标记的抗药基因,以及针对抗药基因的安全性问题而发展的无选择标记的遗传转化技术及生物安全性筛选标记的基因工程技术。葡萄糖胺合成酶基因是近年发展起来的新型生物安全性筛选标记,它弥补了其他营养缺陷互补型和功能附加型筛选标记的缺陷,具有广阔的应用前景。     

Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system

The purpose of this study was to introduce a simple and sensitive plasmid-based noncellular system to evaluate the photoprotection of bacterial melanin on DNA damage against ultraviolet (UV) radiation. Plasmid DNA was used to assess the role of melanin in different ranges of UV using a series of in vitro assays. Fluorometric measurements suggested that melanin could efficiently scavenge reactive oxygen species (ROS) generated by UVA irradiation in solution, and the scavenging capability was proportional to the pigment concentration. The protective effect of melanin on plasmid DNA under UVB irradiation was confirmed by the transformation efficiency of the protected DNA, which was at least 10-fold higher than that of the non melanin protected DNA. After the UVC irradiation, the DNA damage of strand breaks was quantified by laser-induced fluorescence capillary electrophoresis. The percentage of supercoiled plasmid was reduced from 80% to less than 5% without melanin protection. In contrast, the percentage of supercoiled DNA only decreased to about 40% in the presence of melanin under the same radiation conditions. All these results demonstrated that bacterial melanin did protect DNA from being damaged throughout full UV irradiation. This system, avoiding the potential interference by cellular DNA repair machinery and intracellular substances, may provide a sensitive in vitro means to evaluate the functions of melanin and other photoprotective compounds from different sources.     

Engineering of bacterial strains and vectors for the production of plasmid DNA

The demand for plasmid DNA (pDNA) is anticipated to increase significantly as DNA vaccines and non-viral gene therapies enter phase 3 clinical trials and are approved for use. This increased demand, along with renewed interest in pDNA as a therapeutic vector, has motivated research targeting the design of high-yield, cost-effective manufacturing processes. An important aspect of this research is engineering bacterial strains and plasmids that are specifically suited to the production of plasmid biopharmaceuticals. This review will survey recent innovations in strain and vector engineering that aim to improve plasmid stability, enhance product safety, increase yield, and facilitate downstream purification. While these innovations all seek to enhance pDNA production, they can vary in complexity from subtle alterations of the host genome or vector backbone to the investigation of non-traditional host strains for higher pDNA yields.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

Restriction of DNA encoding selectable markers decreases the transformation efficiency of Helicobacter pylori

Helicobacter pylori populations recovered from the human stomach display extensive recombination and quasispecies development, and this suggests frequent exchange of DNA between different strains in vivo. In vitro, however, most H. pylori strains display restriction to the uptake of non-self DNA, as measured using selectable markers, regardless of their competency for transformation with self DNA. We have examined the effect of different selectable markers on double-crossover recombination efficiencies in three reference strains (1061, 26695 & SS1) and one clinical isolate (CHP1) of H. pylori. All strains were efficiently transformable to kanamycin or chloramphenicol resistance by using self-genomic DNA from isogenic mutants bearing the aphA3 or cat cassettes, respectively. However, strains 26695 and CHP1 showed a 3-5-log reduction in transformation efficiency by non-self recombinant DNA containing aphA3, when compared to cat. Strain 1061 readily accepted either cassette, and strain SS1 was poorly tolerant of any non-self DNA. Genome-wide random mutagenesis of these strains was only achievable with a selectable marker that allowed high transformation efficiency. Digestion of 32P-labelled cassettes by H. pylori lysates mirrored the transformation results and indicated that in some strains these cassettes are the targets of enzymatic restriction.     

A radiation target method for size determination of supercoiled plasmid DNA

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.     

A novel antibiotic free plasmid selection system: advances in safe and efficient DNA therapy

The presence of antibiotic resistance genes in the delivered plasmids is one of the drawbacks of modern gene therapy and DNA vaccine applications. Here, we describe a strategy that allows for plasmid selection in bacterial hosts, without the requirement of any selection marker. Several bacterial strains were modified, so that the plasmid's replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction. An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression. Only in the presence of plasmid and, hence, RNA I, was tetR turned down and murA expressed. Different commercially available plasmids could be selected by various modified Escherichia coli strains. We further designed a minimalistic plasmid devoid of any selection marker. All of the clones (n=6) examined, when the modified strain JM109-murselect was used for selection, contained plasmids. Thus, we have designed bacterial host strains that for the first time serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid. Consequently, such plasmids may not only be safer, but due to their decreased size, advantages for the manufacturer and higher transfection efficiencies are anticipated.     

简单快速分离无RNA的大肠杆菌质粒DNA的方法

本文介绍一种简单快速分离质粒ＤＮＡ方法。此方法有两个主要步骤。用这种方法分离的质粒ＤＮＡ纯度高、无ＲＮＡ,并可用于酶切、连接等操作。     

A point mutation in the Medicago sativa GSA gene provides a novel, efficient, selectable marker for plant genetic engineering

Bacterial selectable marker genes (SMG) conferring antibiotic resistance are valuable tools in plant genetic engineering, but public concern and regulatory requirements have stimulated the development of alternative selection systems. We have previously demonstrated that a mutated Synechococcus elongatus HemL gene encoding glutamate 1-semialdehyde aminotransferase (GSA) is an efficient SMG in alfalfa. In fact, GSA is irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is gabaculine insensitive. With the aim to develop a plant derived SMG, we cloned and sequenced the Medicago sativa GSA cDNA and reproduced one of the two mutations associated with gabaculine resistance in Synechococcus, a transversion resulting in a methionine to isoleucine (M → I) substitution. This mutated gene was assessed as a SMG in tobacco and alfalfa Agrobacterium transformation, in comparison with the wild type gene. In tobacco, about 43% of the leaf explants produced green shoots, whereas in alfalfa 47% of the explants produced green embryos in the presence of 30 μM gabaculine when the M → I GSA was introduced. Escapes were absent in tobacco and only 6% in alfalfa. No effect on the plant phenotype was noticed. We propose this new SMG as a widely acceptable alternative to those currently used.     

Rational Vector Design for Efficient Non-viral Gene Delivery: Challenges Facing the Use of Plasmid DNA

Although non-viral gene delivery is a very straightforward technology, there are currently no FDA-approved gene medicinal products available. Therefore, improving potency, safety, and efficiency of current plasmid DNA vectors will be a major task for the near future. This article will provide an overview on factors influencing production yield and quality as well as safety issues that emerge from the vector design itself. Special focus will be on generating bacterial pDNA vectors by circumventing the use of antibiotic resistance genes, to generate safer gene medicinal products as well as smaller, more efficient DNA vectors.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

一种提取质粒DNA的改良方法

本文详细介绍了一种改良碱裂解法提取质粒ＤＮＡ的方法,该法采用ＮＨ４Ａｃ代替苯酚和氯份的抽提过程,得率高,质量好,完全达到了分子生物学常规实验的要求,如酶切、连接、转化大肠杆菌、ＰＣＲ等,甚至用于序列测定和植物遗传转化,该法重复性好,操作简单、实用．