Early events in microprojectile bombardment: cell viability and particle location

Tungsten and gold particles, coated with plasmid DNA harboring the β-glucuronidase (GUS) and neomycin phosphotransferase II (npt-II) genes, were delivered into tobacco primary leaves and suspension-cultured cells of maize using the helium particle inflow gun. Cell viability and particle localization were determined 1 and 2 days after bombardment. Of the counted particles, 7–10% penetrated into or through the epidermis. Blue spots on tobacco leaves appeared as a blue area around a single, densely stained particle-containing central cell. DNA-coated gold particles provoked smaller spots with less diffusion and gave rise to more individual events than tungsten particles. In more than 90% of the GUS-positive epidermal and mesophyll cells, a particle was detectable within their nucleus. Two days after bombardment, viability had decreased to 1–2% in particle-containing cells. Penetration of a cell by a particle was accompanied by callose formation in the wound area. Dead suspension culture cells of maize without callose formation but containing particles were detected just 1 h post-bombardment. Living cells with callose spots appeared more frequently after bombardment with tungsten than gold. As in tobacco, GUS expression was limited to those cells containing a particle in their nucleus, and the number of particle-containing, viable cells was low after 48 h. The frequency of stable expression events was compared to the number of surviving tobacco leaf cells. On average, four kanamycin-resistant calli or plantlets were recovered per bombarded dish, of which approximately 50% were also GUS-positive. This corresponds to a stable-to-transient ratio of approximately 0.8%, and is similar to the number of particle-containing cells surviving after 48 h.     

Factors influencing transient gene expression in bean (Phaseolus vulgaris L.) using an electrical particle acceleration device

Summary The parameters influencing transient expression of the betaglucuronidase gene in bean embryonic axes, cotyledons, apical meristems and leaves were evaluated after gene delivery with an electrical particle acceleration device. A calciumspermidine procedure for coating gold particles with DNA resulted in higher levels of GUS expression with lower concentrations of gold particles compared with a calcium phosphate procedure. The DNA concentration, distance between the discharge chamber and the retaining screen and the vacuum in the apparatus also influenced gene delivery. Sections prepared for light and electron microscopy showed the localisation, within target cells, of gold particles used to deliver the DNA. Immunolocalization of foreign gene expression within cells confirmed an even distribution of gene product throughout the cell cytoplasm.Abbreviations BAP 6,benzylaminopurine - BSA bovine serum albumin - MS Murashige and Skoog (1962)     

Effects of microprojectile bombardment on embryogenic suspension cell cultures of maize (Zea mays L.) used for genetic transformation

We have investigated the interaction between tungsten and gold microprojectiles with suspension-culture cells of maize used for genetic transformation. Particle size measurements were evaluated before and after DNA precipitation to determine mean particle size and the effect of DNA precipitation on particle aggregation. Following particle bombardment, metal foils were examined by scanning electron microscopy to visualize dispersion of individual particles and aggregates. Particle penetration into suspension-culture cell clusters was examined in paraffin-embedded bombarded cells serially sectioned and viewed with light microscopy and by energy dispersive X-ray microanalysis. Acridine-orange-stained bombarded cells were examined to observe cellular response to particle penetration. Transient expression of reporter genes C1 and B and GUS, (-glucuronidase) were used to assess effects of particle bombardment on embryogenic cell types. Autoradiographic analysis of the transformable suspension cell culture SC82 (see Gordon-Kamm et al. 1990, Plant Cell 2, 603–618) was conducted to evaluate the S-phase and mitotic indices in embryogenic and nonembryogenic cells throughout a subculture passage and in response to DNA/particle delivery. The results of these investigations are discussed relative to cytodifferentiation of suspension cell clusters and recovery of transformed clonal sectors.Abbreviations GUS -glucuronidase - FAA formaldehyde-acetic acid-alcohol - SEM scanning electron microscopy     

A quantitative and histological comparison of gus expression with different promoter constructs used in microprojectile bombardment of peanut leaf tissue

Summary β-glucuronidase (GUS) expression driven with different promoter constructs was quantitatively and histologically compared in peanut leaf tissue following microprojectile bombardment. X-Gluc staining patterns varied with the construct used. Tissues bombarded with the pAC2GUS construct had larger foci and a greater percentage of area staining blue. pEmuGN exhibited the greatest numbers of blue spots compared to pAC2GUS and pTRA140. Histological evaluations of blue staining foci showed a diffusion gradient of blue precipitate from a central, prominently-staining cell outward to as many as seven cell layers. The intensity of X-gluc product in centrally-staining cells varied. Gold microprojectile particles were usually located within the three surface cell layers. Depending on the plasmid construct, 72–90% of the centrally-staining cells had at least one gold particle. However, the presence of GUS expression did not appear to require a microprojectile within the nucleus, which was observed in 37% or fewer of the centrally-staining cells. With the pAC2GUS construct, staining patterns varied with location within leaflets and had an “edge effect,” i.e., blue spots were frequently larger at the margin versus central regions. This enhanced activity could be anticipated with an actin promoter in the more mitotically active marginal leaf cells. Total GUS activity as determined by fluorometric analyses was correlated with the percentage of X-gluc stained area. The pAC2GUS construct exhibited the highest total GUS activity among the three constructs.     

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.     

Optimisation of procedures for microprojectile bombardment of microspore-derived embryos in wheat

Using the PDS-1000/He Biolistic® Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term β-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five μl pAHC25 (0.75 mg ml^-1 in TE buffer) was precipitated onto 25 μl gold particles (60 mg ml^-1 in sterile water), using 20 μl spermidine (0.1 M) and 50 μl CaCl₂ (2.5 M). The particles were centrifuged and resuspended in 75 μl absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29′′ Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 ± 17 GUS-positive foci/MDE (mean ± s.e.m, n=3), with 17 ± 4 foci/MDE at 15 days, giving a 3.0-fold increase (p<0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.     

Transfection by particle bombardment: Delivery of plasmid DNA into mammalian cells using gene gun

Background

Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment.

Methods

gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay.

Results

This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles.

Conclusions

This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 μm gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 μg DNA/mg gold particles.

General significance

These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination.     

A rapid method to monitor DNA precipitation onto microcarriers before particle bombardment

The binding or precipitation of DNA onto gold or tungsten microcarriers represents one of the most crucial steps for gene transfer via the particle bombardment process. We have developed a simple and rapid method to monitor DNA precipitation onto microcarriers before delivery to intact cells or tissues. Binding of DNA constructs to different microcarriers was evaluated with relative fluorescence values using a dedicated fluorometer. Significantly greater precipitation was detected using gold vs. tungsten microcarriers. Addition of glycerol resulted in a 46% increase in precipitation. A 42% difference in precipitation was observed using two different brands of polyproplyene tubes. Fluorescence values dropped 10–50% 3 hr after initial precipitation. Fluorescence values were correlated with the number of transient GUS transformants of rice (Oryza sativa, L.) cells. Precipitation with PEG gave higher fluorescent values and GUS transformants than a similar method without PEG. Results from these experiments indicate that fluorescence measurements are an effective and rapid method to monitor DNA precipitation for particle bombardment experiments.Communicated by C. Quiros     

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica,and Peony (Paeonia lactiflora) by Particle Bombardment

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.     

β-Glucuronidase gene and green fluorescent protein gene expression in de-exined pollen of Nicotiana tabacum by microprojectile bombardment

 Gene constructs containing the β-glucuronidase (GUS) gene or green fluorescent protein (GFP) gene under the control of pollen-specific promoter Zm13-260 from maize were introduced by particle bombardment into de-exined pollen of Nicotiana tabacum. The de-exined pollen exhibited transient expression of the GUS or GFP gene as indicated by histochemical and fluorescent assay, respectively. The frequency of de-exined pollen transformation with the GUS or GFP gene was approximately 6 and 3 times higher, respectively, than that of pollen with intact walls, indicating that pollen deprived of the exine barrier responded better to foreign gene transfer than did the original. Cytological observation of GUS-expressing pollen grains showed that introduced gold particles were visible in the cytoplasm and vegetative nucleus as well as in the generative nucleus. GFP-expressing pollen tubes were observed in the style even after pollination. Received: 28 October 1997 / Revision accepted: 13 April 1998     

基因枪法将GAI基因导入巴西橡胶的研究

为了探讨利用基因工程技术进行橡胶树品质改良的可行性,用基因枪轰击巴西橡胶(Hevea brasiliensis)愈伤组织,将GAI矮化基因导入橡胶受体中,通过50mg L-1卡那霉素筛选鉴定,优化了基因枪法转化橡胶的各种参数。结果表明,DNA金弹与靶细胞的距离为9cm,每皿轰击一次时,胚状体诱导率可以达到1.87%。经过GUS组织染色和PCR扩增鉴定,初步确定GAI基因已经整合到橡胶基因组中。     

Analysis of particle bombardment parameters to optimise DNA delivery into wheat tissues

 The objective of this study was to identify the major parameters controlling DNA delivery by particle bombardment to wheat (Triticum aestivum L.) scutellum and inflorescence tissue. The main factors studied were the DNA/gold precipitation process, bombardment parameters and tissue culture variables. Efficiency of DNA (uidA gene) delivery was assessed by scoring transient GUS expression in bombarded tissues. Of the parameters analysed, amount of plasmid DNA, spermidine concentration, presence of Ca⁺⁺ ions, calcium chloride concentration, amount of gold particles, gold particle size, acceleration pressure, chamber vacuum pressure, bombardment distance, osmotic conditioning of tissues and type of auxin had a clear influence on transient gene expression. A bombardment procedure suitable for elite wheat varieties was developed which allowed high-efficiency DNA delivery combined with reduced damage to target tissues. Received: 6 May 1998 / Revision received: 10 August 1998 / Accepted: 2 February 1999     

Laser Plasma Jet Driven Microparticles for DNA/Drug Delivery

This paper describes a microparticle delivery device that generates a plasma jet through laser ablation of a thin metal foil and uses the jet to accomplish particle delivery into soft living targets for transferring biological agents. Pure gold microparticles of 1 µm size were coated with a plasmid DNA, pIG121Hm, and were deposited as a thin layer on one surface of an aluminum foil. The laser (Nd:YAG, 1064 nm wavelength) ablation of the foil generated a plasma jet that carried the DNA coated particles into the living onion cells. The particles could effectively penetrate the target cells and disseminate the DNA, effecting the transfection of the cells. Generation of the plasma jet on laser ablation of the foil and its role as a carrier of microparticles was visualized using a high-speed video camera, Shimadzu HPV-1, at a frame rate of 500 kfps (2 µs interframe interval) in a shadowgraph optical set-up. The particle speed could be measured from the visualized images, which was about 770 m/s initially, increased to a magnitude of 1320 m/s, and after a quasi-steady state over a distance of 10 mm with an average magnitude of 1100 m/s, started declining, which typically is the trend of a high-speed, pulsed, compressible jet. Aluminum launch pad (for the particles) was used in the present study to make the procedure cost-effective, whereas the guided, biocompatible launch pads made of gold, silver or titanium can be used in the device during the actual clinical operations. The particle delivery device has a potential to have a miniature form and can be an effective, hand-held drug/DNA delivery device for biological applications.     

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with -glucuronidase (GUS) mRNA with a 5 cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.     

Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter β-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula × P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation. Received: 24 April 1997 / Accepted: 17 July 1997     

THE EFFECT OF THE ELECTRON-OPAQUE PORE MATERIAL ON EXCHANGES THROUGH THE NUCLEAR ANNULI

To investigate the extent to which the electron-opaque pore material can regulate nucleocytoplasmic exchanges which occur through the nuclear annuli, experiments were performed in which polyvinylpyrrolidone (PVP)-coated colloidal gold particles (25 to 170 A in diameter) were microinjected into the cytoplasm of amebas (Amoeba proteus). The cells were fixed at various times after injection and examined with the electron microscope in order to determine the location of the gold particles. High concentrations of gold were found associated with the pore material at specific points adjacent to and within the pores. It is tentatively suggested that such specific accumulations could be a means of selecting substances from the cytoplasm for transport through the pores. Particles were also scattered throughout the ground cytoplasm and nucleoplasm. A comparison of the diameters of particles located in these two regions showed that the ability of materials to penetrate the nuclear envelope is a function of their size. It was estimated that the maximum size of the particles able to enter the nucleus is approximately 125 to 145 A indiameter. The regulation of exchanges with regard to particle size is thought to be dependent on the specific organization of the electron-opaque pore material.     

Optimization of transient gene expression in pollen of Norway spruce (Picea abies) by particle acceleration

In order to optimize transient gene expression in Norway spruce pollen after DNA delivery with particle bombardment, effects of different conditions during homhardmenl were analysed using β-glucuroniduse (GUS) driven by the rice Act I promoter and Inciferase (LUS) driven by the tomato !at 52 promoter as reporter genes. Transient gene expression was significantly increased hy using two bombardments. Also the distance from the stopping plate to the sample was critical to gam maximum gene expression. There was no significant difference between gold and tungsten particles, and the number of positively stained pollen increased with increasing DNA concentration, from 5 to 40 pg DNA added in the DNA/tungsten solution The DNA delivery to Norway spruce pollen was most efficient at a chamber pressure above 70 kPa.     

Transformation of wheat (Triticum aestivum L.) microspore-derived callus and microspores by particle bombardment

Twenty-seven bialaphos-tolerant and GUS-positive lines were produced from 2,940 callus pieces after particle bombardment of wheat microspore-derived callus. Regenerated plants were mainly of the albino type. In an attempt to avoid this problem, wheat microspores were used as target cells for particle bombardment. Pre-cultivation for a period of 3-8 days improved the frequency of GUS-expressing microspores. Helium rupture pressures between 1,100 psi and 1,800 psi, the amount of gold per bombardment (ranging from 37 µg to 300 µg) and particle size (0.6-1.0 µg) did not significantly affect transient expression. Microspore response measured as number of recovered embryos was not significantly affected by variations in helium pressure or amount of gold used, but response was significantly influenced by particle size. The highest number of GUS-expressing embryos was 3.5 embryos per 10⁶ microspores, which was obtained after 4 days of pre-cultivation, 1,350 psi rupture pressure, 0.6+1.0 µm particles (1:1) and 150 µg gold particles per bombardment.     

Erratum to: Transformation of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) microspore-derived callus and microspores by particle bombardment

Twenty-seven bialaphos-tolerant and GUS-positive lines were produced from 2,940 callus pieces after particle bombardment of wheat microspore-derived callus. Regenerated plants were mainly of the albino type. In an attempt to avoid this problem, wheat microspores were used as target cells for particle bombardment. Pre-cultivation for a period of 3-8 days improved the frequency of GUS-expressing microspores. Helium rupture pressures between 7,584 kPa and 12,411 kPa, the amount of gold per bombardment (ranging from 37 µg to 300 µg) and particle size (0.6-1.0 µm) did not significantly affect transient expression. Microspore response measured as number of recovered embryos was not significantly affected by variations in helium pressure or amount of gold used, but response was significantly influenced by particle size. The highest number of GUS-expressing embryos was 3.5 embryos per 10⁶ microspores, which was obtained after 4 days of pre-cultivation, 9,308 kPa rupture pressure, 0.6+1.0 µm particles (1:1) and 150 µg gold particles per bombardment.