Physical trauma and tungsten toxicity reduce the efficiency of biolistic transformation

A cell suspension culture of tobacco (Nicotiana tabacum L.) was used as a model to study injury to cells during biolistic transformation. Lawns of cells were bombarded with tungsten particles that were coated with a plasmid containing the β-glucuronidase and the neomycin phosphotransferase II genes. When a gunpowder-driven biolistic device was used, numerous transiently expressing cells were focused around the epicenter of the blast which was manifested by a hole blown in the filter paper supporting the cells. However, transformed cells nearest the blast epicenter were injured and could not be recovered as stable transformants. The injury was primarily caused by physical trauma to the cells from gas blast and acoustic shock generated by the device. Postlaunch baffles or meshes placed in the gunpowder device reduced cell injury and increased the recovery of kanamycin-resistant colonies 3.5- and 2.5-fold, respectively. A newly developed helium-driven device was more gentle to the cells and also increased the number of transformants. Cell injury could be further moderated by using a mesh and a prelaunch baffle in the helium device. Toxicity of the tungsten microprojectiles also contributed to cell injury. Gold microprojectiles were not toxic and resulted in fourfold more kanamycin-resistant colonies than when similar quantities of similarly sized tungsten particles were used.     

An Efficient Rice Transformation System Utilizing Mature Seed-derived Explants and a Portable,Inexpensive Particle Bombardment Device

We developed a practical and efficient gene transfer system for indica rice utilizing mature-seed derived explants and a simple bombardment device which uses compressed helium for accelerating DNA-coated metal particles. Unlike instruments which have been described in the literature previously, this new bombardment device, which is an improvement of the particle inflow concept, does not require vacuum. This attribute simplifies the transformation procedure significantly and it makes rice transformation technology accessible to laboratories which may not have the resources to invest in more expensive particle bombardment instruments. We determined experimentally that we could recover transgenic rice plants utilizing three different particle bombardment instruments at comparable frequencies.     

Development of the particle inflow gun for DNA delivery to plant cells

Summary A simple and inexpensive particle bombardment device was constructed for delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles using pressurized helium in combination with a partial vacuum. The particles are accelerated directly in a helium stream rather than being supported by a macrocarrier. Bombardment parameters were partially optimized using transient expression assays of a ß-glucuronidase gene in maize embryogenic suspension culture and cowpea leaf tissues. High levels of transient expression of the ß-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of corn and soybean, and leaf tissue of cowpea. Stable transformation of embryogenic tissue of soybean has also been obtained using this bombardment apparatus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - GUS ß-glucuronidase - NOS nopaline synthase Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark of proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 34-92     

Biolistic transformation of Trichoderma reesei using the Bio-Rad seven barrels Hepta Adaptor system

Effective biolistic transformation of intact conidia from the filamentous fungus Trichoderma reesei was achieved using the Bio-Rad Hepta Adaptor system with seven barrels for particle launch. Transformation frequencies of up to 39 colonies per microg of circular DNA and 37 colonies per microg of linear DNA were obtained at an optimal target distance of 3 cm and a helium pressure of 1350 psi. These values are about 3.5- to 6-fold higher than transformant yields reported earlier for T. reesei using the hygromycin phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin B as a selectable marker in combination with the PDS-1000/He single barrel system. High mitotic stability of the transformants (98-100%) was demonstrated. The Hepta Adaptor device allowing bombardment of seven lots of conidia in a single plate offers clear advantage in terms of transformant numbers over the single barrel system where target cells are restricted to the center of the plate.     

The possible use of PKZh-type devices (liquid purity control devices) in the analysis of cell suspensions

PKZh means "device for liquid purity control". A possibility is considered to use the native PKZh type device for carrying out quantitative analyses of cellular suspension components, for routine bacterial suspension, agglutinated bacterial suspension and erythrocyte suspension. The flowing photometric principle of particle recording, used in the device, allows to analyse biological suspensions with small amounts of components. The device provides a differential count of some cells and their conglomerates in six dimensional ranges, within the frames of 1-25 micron or higher. The time consumption for one sample analysis is 10-15 seconds.     

Technical advance: an automated device for cryofixation of specimens of electron microscopy using liquid helium

Metal-contact rapid freezing using liquid helium is theoretically the best method for preserving the fine structure of living cells with high temporal resolution in preparation of tissue samples for electron microscopy. However, this method is not commonly used, because of its technical difficulty and low reproducibility. We have designed and constructed an automatic device which allows simple, rapid and reproducible preparation of high-quality electron microscopic specimens by the non-specialist. We assessed the quality of cryofixation in samples prepared using this device by examining the preservation of cellular ultrastructure in relation to distance from the freezing block, and found that the region within 10 microm of the metal-contact plane was fixed with the highest quality. We applied this device, in combination with freeze-substitution methods and immunocytochemical techniques, to two phenomena involving rapid movement of subcellular components: (1) active movement of subcellular structures in the papillar cells of stigma and (2) light-induced rapid subcellular translocation of phytochrome A. Considering the importance of understanding subcellular processes of living cells for molecular and cell biology, this device will be a useful tool for diverse biological applications in the near future.     

Ploidy of a mutant Saccharomyces cerevisiae defective in homologous recombination

The ploidy of a mutant of Saccharomyces cerevisiae defective in recombination (Rec-) has been determined using tetrad analysis and flowing fluorometry. Evidence is obtained that the effect of the Rec- phenotype, i.e. the increase of the stability of plasmids with 2 mkm DNA ori replication in the yeast cirO cells is not the result of the diploidy in cells of the Rec- mutant developed in the process of transformation.     

Capturing cancer cells using aptamer-immobilized square capillary channels

We report a simple square capillary-based cell affinity chromatography device that utilizes a coating of aptamers for selective capture of target cancer cells from a flowing suspension. The device consists of a square capillary with an inner diameter of roughly five cell diameters, connected via Teflon tubing to a syringe. Aptamers are immobilized on the inner surface of the capillary through biotin-avidin chemistry, the extent of which can be controlled by adjusting the aptamer concentration. Introduction of different cell types into separate devices, as well as mixtures of target and non-target cells, demonstrated that aptamer-target cells can be captured in significantly higher concentrations compared to non-target cells. Once optimized, 91.1 ± 3.5% capture efficiency of target leukemia cells was reported, as well as 97.2 ± 2.8% and 83.6 ± 5.8% for two different colon cancer cell lines. In addition, cells captured in the device were imaged, and the square capillary exhibited better optical properties than standard cylindrical capillaries, leading to the detection of leukemia cells in blood samples. Compared to current microfluidic cell affinity devices, this capture device requires no complicated design or fabrication steps. By providing a simple means of detecting and imaging cancer cells in the blood, this work has potential to directly assist clinicians in determining disease prognosis and measuring therapeutic response.     

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.     

Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles

Summary We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.Abbreviations BMS Black Mexican Sweet - RPM revolutions per minute - uidA -glucuronidase gene - GUS -glucuronidase protein - LB Luria-Bertani broth - OD600 optical density at 600 nm - psi pounds per square inch - Ap^r ampicillin resistance - Kn^r kanamycinresistance     

Transformation of elite white maize using the particle inflow gun and detailed analysis of a low-copy integration event

Elite white maize lines W506 and M37W were transformed with a selectable marker gene (bar) and a reporter gene (uidA) or the polygalacturonase-inhibiting protein (pgip) gene after bombardment of cultured immature zygotic embryos using the particle inflow gun. Successful transformation with this device did not require a narrow range of parameters, since transformants were obtained from a wide range of treatments, namely pre-culture of the embryos for 4-6 days, bombardment at helium pressures of 700-900 kPa, selection-free culture for 2-4 days after bombardment and selection on medium containing bialaphos at 0.5-2 mg l^-1. However, bombardments with helium pressures below 700 kPa yielded no transformants. The culture of immature zygotic embryos of selected elite white maize lines on medium containing 2 mg l^-1 2,4-dichlorophenoxyacetic acid and 20 mM L-proline proved to be most successful for the production of regenerable embryogenic calli and for the selection of putative transgenic calli on bialaphos-containing medium after transformation. Transgenic plants were obtained from four independent transformation events as confirmed by Southern blot analysis. Transmission of the bar and uidA genes to the T₄ progeny of one of these transformation events was demonstrated by Southern blot analysis and by transgene expression. In this event, the transgenes bar and uidA were inserted in tandem.     

A low-pressure gene gun for genetic transformation of maize (Zea mays L.)

We have successfully used the low-pressure BioWare gene gun, developed for gene transfer in animal cells, for plant tissues. The BioWare device is easy to manipulate. Just 50 psi helium pressure was sufficient to transfer foreign genes into the aleurone layer and embryo of maize without causing tissue damage in the impact area. As shown by expression signals from invasive histochemical β-glucuronidase (GUS) activity, the foreign reporter gene expressed well in bombarded tissues. This successful GUS-transient expression extends the application of this low-pressure gene gun from animal cells to plant tissues.     

Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells.

Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 10(4) per plate per 0.8 micrograms DNA. Although transformation rates for the other species were low (less than 10(2) per plate per 0.8 micrograms DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.     

Development of the Particle Inflow Gun

A simple and inexpensive particle acceleration apparatus was designed for direct delivery of DNA to plant cells. The Particle Inflow Gun (PIG) is based on acceleration of DNA-coated tungsten particles directly in a helium steam. High levels of transient expression of theβ-glucuronidase gene were obtained following bombardment of embryogenic suspension cultures of maize and soybean, and leaf tissue of cowpea. Stable transformation of soybean and maize has also been obtained using this bombardment apparatus.     

Development of an airgun device for particle bombardment

Construction and operation of an airgun device for transient gene-expression studies in monocots is described. Compressed air in a cylinder of an airgun was used as the source of propulsion for DNA-coated gold or tungsten particles. Under a partial vacuum of 700 mm Hg, velocity of the macrocarrier was measured at 520 m sec^?1 and 432 m sec^?1 at atmospheric pressure. Optimum distance from the stopping plate to different target cells during bombardment ranged from 4 to 7 cm. Mean transformation efficiency of the GUS-gene marker was estimated at 350 transformants per 65 mg fresh weight of the maize cultured cells. Up to 200 GUS transformed cells were detected per 100 mg of embryogenic rice callus. Use of gold flakes or tungsten powder as microcarriers resulted in similar transformation rates. No transformation was observed in any cells when DNA constructs contained prokaryotic translation initiation sequences for the GUS gene. Based on transient GUS assays, further modification of the airgun device will likely be necessary to obtain high stable transformation rates.     

The Biolistic® PDS-1000/He device

This paper describes the design, operation, and performance of the Biolistic® PDS-1000/He device, which is used to transform living organisms with foreign DNA. DNA is delivered to cells in association with microscopic metal particles, called microcarriers, that are propelled at high velocity towards target tissues. The microcarriers are accelerated on a plastic cylinder, called a macrocarrier, which is driven by a shock wave of helium gas. The effectiveness of the PDS-1000/He device was tested by bombarding tobacco cell suspension cultures with microcarriers that were coated with plasmid DNA containing the B-glucuronidase (GUS) and neomycin phosphotransferase II (NPTII) genes. Two days after bombardment, there were 6835 ± 594 cell clusters per petri plate that expressed the GUS gene. Kanamycin resistant colonies were observed 6 to 8 weeks after bombardment, at a rate of 838 ± 134 colonies per bombarded plate.     

Optimization of biological and physical parameters for biolistic genetic transformation of common wheat (Triticum aestivum L.) using a particle inflow gun

The parameters for delivery of expression cassettes to cells of wheat morphogenic callus induced from immature embryos were optimized. Three systems (gradation, delayed, and regeneration) for in vitro selection of transgenic wheat tissue using the bar gene, providing resistance to the herbicide phosphinothricin (PPT), were compared. The efficiency of gene delivery to the cells competent for plant regeneration was assessed by comparing the number of spots transiently expressing uidA gene (encoding beta-glucuronidase) per unit surface of the morphogenic calluses treated under various conditions. The selection systems in question were evaluated by comparing the transformation efficiency frequencies. The optimal parameters for wheat biolistic transformation using a particle inflow gun were determined, namely, the distance between the particle source and the target tissue (12 cm) and helium pressure during the shot (6 atm). The optimal time of callus tissue development on the medium inducing callus formation was determined (10-14 days). Comparison of the three selection variants demonstrated that the regeneration system was the most efficient for producing true transgenic plants of common wheat.     

Whole-cell imaging at nanometer resolutions using fast and slow focused helium ions

Observations of the interior structure of cells and subcellular organelles are important steps in unraveling organelle functions. Microscopy using helium ions can play a major role in both surface and subcellular imaging because it can provide subnanometer resolutions at the cell surface for slow helium ions, and fast helium ions can penetrate cells without a significant loss of resolution. Slow (e.g., 10–50 keV) helium ion beams can now be focused to subnanometer dimensions (∼0.25 nm), and keV helium ion microscopy can be used to image the surfaces of cells at high resolutions. Because of the ease of neutralizing the sample charge using a flood electron beam, surface charging effects are minimal and therefore cell surfaces can be imaged without the need for a conducting metallic coating. Fast (MeV) helium ions maintain a straight path as they pass through a cell. Along the ion trajectory, the helium ion undergoes multiple electron collisions, and for each collision a small amount of energy is lost to the scattered electron. By measuring the total energy loss of each MeV helium ion as it passes through the cell, we can construct an energy-loss image that is representative of the mass distribution of the cell. This work paves the way to use ions for whole-cell investigations at nanometer resolutions through structural, elemental (via nuclear elastic backscattering), and fluorescence (via ion induced fluorescence) imaging.     

High throughput particle analysis: combining dielectrophoretic particle focussing with confocal optical detection

A micro flow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 microm high and 250 microm wide. The method ensures that all the particles pass through an interrogation region approximately 5 microm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 microm beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocity of 10 mm s(-1)).     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.