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PubMedDate not supplied

Stable genetic transformation of Eschscholzia californica expressing synthetic green fluorescent proteins.

An efficient protocol is described for the stable genetic transformation of Eschscholzia californica (California poppy) using Agrobacterium tumefaciens as a vector. We have employed the disarmed A. tumefaciens LBA4404 encoding a synthetic green fluorescent protein reporter gene that is further controlled by an enhanced cauliflower mosaic virus 35S promoter. Stably transformed E. californica cells appear 3 weeks after initial cocultivation of A. tumefaciens with poppy leaves, stems, or roots. Transformed poppy calli were visualized by exposure to long-wave UV or blue light and analyzed in detail by fluorescent microscopy and laser-scanning microscopy. Moreover, green fluorescent calli have been maintained through continual subculture and grow well either on Gamborg's B5 agarose or liquid medium.

Agrobacterium tumefaciens↗

PubMed1980

Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid.

A number of heterologous plasmid deoxyribonucleic acids (DNAs) coding for erythromycin, tylosin, lincomycin, tetracycline, or chloramphenicol resistance have been introduced into Streptococcus pneumoniae via genetic transformation with frequencies that varied between 10(-5) to as high as 5 x 10(-1) per colony-forming unit. Transformation with plasmid DNA required pneumococcal competence, was competed by chromosomal DNA, and showed a saturation at about 0.5 micrograms/ml (with a recipient population of 3 x 10(7) colony-forming units of competent cells per ml). Plasmid transformation did not occur with a recipient strain, 410, defective in endonuclease I activity and in chromosomal genetic transformation. All erythromycin-resistant transformants examined contained covalently closed circular DNA with the same electrophoretic mobility on agarose gels as the donor DNAs, and when examined in detail the plasmid reisolated from the transformants had the same restriction patterns and the same specific transforming activity as the donor DNA. In the cases of two plasmids examined in detail--pAM77 and pSA5700 Lc9--most of the transforming activity was associated with DNA monomers; DNA multimers present in pSA5700 Lc9 also had biological activity. An unexpected finding was the demonstration of transformation (2 x 10(-5) per colony-forming unit) with plasmid DNAs linearized by treatment with S1 nuclease or with restriction endonucleases.

Bacillus subtilis↗

PubMed1980

The preparation of transforming DNA from Mycoplasma hominis strain Sprott tetr and quantitative studies of the factors affecting the genetic transformation of Mycoplasma salivarium strain S9 tets to tetracycline resistance.

DNA extracted by a standard method from Mycoplasma hominis Sprott, resistant to 100 micrograms tetracycline, permitted the quantitative genetic transformation of tetracycline-sensitive Mycoplasma salivarium to resistance. The yield was 1 microgram DNA/10(9) cells. This DNA enabled determination of the optimum conditions for making M. Salivarium competent with CaCl2 and for studying some factors affecting transformation. Mycoplasma salivarium was transformed to resistance to 10, 20, and 30 micrograms tetracycline but not to 40 micrograms. The optimum DNA concentration for transforming resistance to 10, 20, and 30 micrograms tetracycline was the same, i.e., 50 micrograms DNA/10(8) viable cells. Treatment with DNase indicated that DNA uptake took 30 min. Competition between transforming DNA and DNA from calf thymus and M. salivarium tets inhibited transformation.

Calcium Chloride↗

PubMed1988

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

Bacteriological Techniques↗

PubMed2006

[Study on establishment of system with high frequency for genetic transformation of Phellodendron chinese and plant regeneration].

OBJECTIVE: To establish an effective system for genetic transformation of Phellodendron chinese by Agrobacterium rhizogenes. METHOD: A. rhizogenes strains R1601 was employed to induce the optimum hairy roots and the hairy roots were developed into regenerated plants on solid MS media with different kinds of plant growth regulators. RESULT: An effective protocol in optimized conditions were the hypocotyl with cotyledon after 10 d cultured, by 36 h for pre-culture, 15 min for infection, 4 d for co-culture and dosage 60 micromol/L AS in culture A. rhizogenes. It was clearly demonstrated that T-DNA of A. rhizogenes Ri plasmid was integrated into the cells of hairy roots and regenerated plants by the experiments of PCR. CONCLUSION: A. rhizogenes strains R1601 can induce hairy roots of Phellodendron chinese in optimized conditions and the obtained hairy roots can develop into regenerated plants.

Acetophenones↗

PubMed2006

In vitro regeneration and Agrobacterium-mediated genetic transformation of Euonymus alatus.

An in vitro plant regeneration method and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Euonymus alatus. More than 60% of cotyledon and 70% of hypocotyl sections from 10-day-old seedlings of E. alatus produced 2-4 shoots on woody plant medium (WPM) supplemented with 5.0 mg/l 6-benzylaminopurine (BA) plus 0.2 mg/l alpha-naphthalene acetic acid (NAA), and 77% of shoots produced roots on WPM medium with 0.3 mg/l NAA and 0.5 mg/l Indole-3-butyricacid (IBA). On infection with Agrobacterium tumefaciens strain EHA105 harboring a gusplus gene that contained a plant recognizable intron from the castor bean catalase gene to ensure plant-specific beta-glucuronidase (GUS) expression, 16% of cotyledon and 15% of hypocotyl explants produced transgenic shoots using kanamycin as a selection agent, and 67% of these shoots rooted. Stable insertion of T-DNA into the host genome was determined with organ- and tissue-specific expression of the gusplus gene and further confirmed with a PCR-based molecular analysis.

Agrobacterium tumefaciens↗

PubMed1997

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius.

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5'-phosphate decarboxylase (URA3) or orotidine-5'-pyrophosphate (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A transformation frequency of approximately 10(4)/micrograms DNA was obtained, which is tenfold higher than results described in either reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid.

Genetic Markers↗

PubMed2006

Stable Agrobacterium-mediated genetic transformation of London plane tree (Platanus acerifolia Willd.).

London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene beta-glucuronidase (GUS), we have followed the transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD(600) of 0.8-1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer. Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established. By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines, the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome.

Agrobacterium tumefaciens↗

PubMedDate not supplied

[Genetically transformed plant roots as a model for studying specific metabolism and symbiotic contacts of the root system].

Genetic transformation of plants mediated by Ri plasmid of Agrobacterium rhizogenes occupies a special place in plant cell engineering, since this technique based on a natural phenomenon allows cultivation of separately growing roots on hormone-free media. Application of wild-type unmodified agrobacterial strains allows us to obtain root cultures capable of long-term growth in vitro due to an increased sensitivity of the cells to auxins while other biochemical properties remain unaltered. A collection of pRi T-DNA transformed roots of certain dicotyledons was constructed; some strains of it are used to study synthesis of secondary metabolites in root cells. The in vitro cultivated roots could synthesize root-specific metabolites, which allows their large-scale application for biotechnological production of ecologically pure crude drugs. Cocultivation of pRi T-DNA transformed roots with arbuscular mycorrhizal fungi makes possible vital study of all stages of obligate symbiont development and interaction with plant roots. Mixed axenic culture of AM fungi and pRi T-DNA transformed plants can be used to construct a collection of the most valuable endomycorrhizal fungal species and to produce considerable quantities of homogeneous fungal inoculums.

Coculture Techniques↗

PubMed1986

High-efficiency genetic transformation of maize by a mixture of pollen and exogenous DNA.

High-efficiency genetic transformation was induced in a genetic stock of maize, Zea mays Linnaeus, by self-pollination of the recipient plants along with DNA of the donor. The highest frequency of transformed endosperm per ear was 9.29%. DNA was applied onto silks in a pollen/DNA pasty mixture. The exogenous DNA transferred into endosperm expressed itself in endosperm formation. It is not known, however, whether the exogenous DNA segment in endosperm had been incorporated or whether it was present in the nucleus additionally, fragmentally, or in some other state. It was revealed that all of four possible cases had occurred as follows: exogenous DNA was transferred into (i) both embryo and endosperm of the same kernel, (ii) embryo only, (iii) endosperm only, and (iv) neither embryo nor endosperm. It was also revealed that exogenous DNA that had entered into the embryo had been maintained through embryo formation, germination, vegetative growth and differentiation, and reproductive growth, and finally was manifested in embryo and/or endosperm of the following generation and acted in endosperm formation. The frequency of transformed endosperm in this generation, however, was rather low. It may be that exogenous DNA taken into the embryo was unstable.

Journal Article↗

PubMed2001

Genetic transformation systems in insects.

The past 5 years have witnessed the emergence of techniques that permit the stable genetic transformation of a number of non-drosophilid insect species. These transposable-element-based strategies, together with virus-based techniques that allow the expression of genes to be quickly examined in insects, provide insect scientists with a first generation of genetic tools that can begin to be harnessed to further increase our understanding of gene function and regulation in insects. We review and compare the characteristics of these gene transfer systems and conclude that, although significant progress has been made, these systems still do not meet the requirements of robust genetic tools. We also review risk assessment issues arising from the generation and probable release of genetically engineered insects.

Animals↗

PubMed2001

Particle-inflow-gun-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.): optimizing biological and physical parameters.

The present study was conducted to optimize various biological and physical parameters for developing an efficient and reproducible gene transfer method for genetic transformation of buffel grass. Transformation was carried out using a helium-driven particle inflow gun (PIG). Embryogenic calli produced from mature seeds of buffel grass cv. CC-119 were separately bombarded with four plasmids, containing Actin (pAct1DX), Ubiquitin (pAHC-25; pAHC-27) and CaMV-35S (pCaMVGUS) promoters, coated on tungsten and gold particles. The efficiency of transformation was monitored through transient GUS expression. Different parameters, viz., the type of promoter, type and size of microcarrier, helium gas pressure, distance and time of bombardment, were standardized for delivering DNA into embryogenic calli. Bombardment with plasmid DNA carrying the actin promoter coated on 1.6 micro gold particles, at a helium pressure of 4 bars, a distance of 10 cm for 10 micro sec and 28 mm Hg vacuum in the chamber, produced the best result in transient GUS expression. The Actin promoter was found to be more efficient in driving expression of the GUS gene in buffel grass, followed by Ubiquitin and CaMV-35S promoters. Lower helium pressure was found to be sub-optimal, while higher pressure produced a smaller number of blue spots, probably due to excessive damage to the cells. Maximum of 385 blue spots was observed with gold particles of 1.6 micro size, whereas only 213 blue spots were recorded for tungsten particles of 1.0 micro size. The optimized parameters can be employed for genetic transformation of buffel grass with genes of agronomic importance.

Journal Article↗

PubMed1965

UNSTABLE GENETIC TRANSFORMATION IN BACILLUS SUBTILIS AND THE MODE OF INHERITANCE IN UNSTABLE CLONES.

Iyer, V. N. (Canada Department of Agriculture, Ottawa, Ont., Canada). Unstable genetic transformation in Bacillus subtilis and the mode of inheritance in unstable clones. J. Bacteriol. 90:495-503. 1965.-An ultraviolet-induced mutant has been isolated from the double auxotroph of Bacillus subtilis (bearing the linked mutations try(2) and his(2)). This mutant has the property of yielding unstable transformants which, in the case of transformation to prototrophy, can be recognized by reduced colony size on minimal agar. Unstable transformant clones usually become stabilized early during the development of the clone (within 12 generations). Analyses of genotypes emerging from such clones on stabilization indicate that in some of these clones the exogenote is transmitted linearly; its integrity is sufficiently preserved that it can participate in two independent recombination events in two different cells. Genetic markers on the exogenote are expressed in situations where genetic integration has not occurred.

Bacillus subtilis↗

PubMed2005

Establishing genetic transformation for comparative developmental studies in the crustacean Parhyale hawaiensis.

The amphipod crustacean Parhyale hawaiensis has been put forward as an attractive organism for evolutionary developmental comparisons, and considerable effort is being invested in isolating developmental genes and studying their expression patterns in this species. The scope of these studies could be significantly expanded by establishing means for genetic manipulation that would enable direct studies of gene functions to be carried out in this species. Here, we report the use of the Minos transposable element for the genetic transformation of P. hawaiensis. Transformed amphipods can be obtained from approximately 30% of surviving individuals injected with both a Minos element carrying the 3xP3-DsRed fluorescent marker and with mRNA encoding the Minos transposase. Integral copies of the transposon are inserted into the host genome and are stably inherited through successive generations. We have used reporter constructs to identify a muscle-specific regulatory element from Parhyale, demonstrating that this transformation vector can be used to test the activity of cis-regulatory elements in this species. The relatively high efficiency of this transgenic methodology opens new opportunities for the direct study of cis-regulatory elements and gene functions in Parhyale, allowing functional studies to be carried out beyond previously established model systems in insects.

Amphipoda↗

PubMed1987

Modulation of competence for genetic transformation in Streptococcus pneumoniae.

The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6.8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.

DNA, Bacterial↗

PubMed2006

Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens.

A novel genetic transformation method for barley (Hordeum vulgare L.), based on infection of androgenetic pollen cultures with Agrobacterium tumefaciens, is presented. Winter-type barley cv. 'Igri' was amenable to stable integration of transgenes mediated by A. tumefaciens strain LBA4404 harbouring a vector system that confers hypervirulence, or by the non-hypervirulent strain GV3101 with a standard binary vector. The efficacy of gene transfer was substantially influenced by pollen pre-culture time, choice of Agrobacterium strain and vector system, Agrobacterium population density, medium pH and the concentrations of acetosyringone, CaCl(2) and glutamine. After co-culture, rapid removal of viable agrobacteria was crucial for subsequent development of the pollen culture. To this end, the growth of agrobacteria was suppressed by the concerted effects of appropriate antibiotics, low pH, reduced level of glutamine and high concentrations of CaCl(2) and acetosyringone. Following infection with LBA4404 and GV3101, about 31% and 69%, respectively, of the primary transgenic (T(0)) plants carried a single copy of the sequence integrated. The use of hypervirulent A. tumefaciens and hygromycin resistance as a selectable marker resulted in 3.7 T(0) plants per donor spike. About 60% of the primary transgenic plants set seed, indicating spontaneous genome doubling. An analysis of 20 T(1) populations revealed that four progenies did not segregate for reporter gene expression. This indicates that the approach pursued enables the generation of instantly homozygous primary transgenic plants. The method established will be a valuable tool in functional genomics as well as for the biotechnological improvement of barley.

Agrobacterium tumefaciens↗

PubMed2001

Genetic transformation of HeLa cells by Agrobacterium.

Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.

Agrobacterium tumefaciens↗

PubMed2001

Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes.

Recombinant mammalian glycoproteins produced by the baculovirus-insect cell expression system usually do not have structurally authentic glycans. One reason for this limitation is the virtual absence in insect cells of certain glycosyltransferases, which are required for the biosynthesis of complex, terminally sialylated glycoproteins by mammalian cells. In this study, we genetically transformed insect cells with mammalian beta 1,4-galactosyltransferase and alpha 2,6-sialyltransferase genes. This produced a new insect cell line that can express both genes, serve as hosts for baculovirus infection, and produce foreign glycoproteins with terminally sialylated N-glycans.

Animals↗