Evidence That More than 90% of beta-Glucuronidase-Expressing Cells after Particle Bombardment Directly Receive the Foreign Gene in their Nucleus

Plasmid DNA harboring the β-glucuronidase (GUS) gene, coated on gold particles, was delivered into cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells using a pneumatic particle gun. Cytological analyses of intracellular location of the introduced gold particles before and after GUS expression assay indicated that more than 90% of GUS-expressing cells after bombardment received a DNA-coated particle in their nucleus.     

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.     

Evaluation of parameters for high efficiency gene transfer via particle bombardment in Indian mulberry

Particle bombardment is a popular method of direct gene delivery into cell, tissue and organs since it requires minimum pre- and post-bombardment manipulation. In addition, this technique is much easier and fast to perform with intact tissue/organ and reduces the period of in vitro culture. Genetic transformation of mulberry, Morus indica cv. K2 was attempted by particle bombardment using hypocotyl, cotyledon, leaf and leaf callus explants. The effect of various physical and biological parameters during bombardment were studied by the histochemical localization of GUS reporter gene following two days of bombardment and by assessing the number of blue spots per explant. p35SGUSINT was used for optimization of different parameters. The percentage of GUS positive explants was very low with tungsten (20%) as compared to gold particles (36%) indicating tungsten toxicity to the tissue. Maximum GUS activity was observed at 1100 psi helium pressure and 9 cm target distance for hypocotyl, cotyledon and leaf. Double bombardment of explants with 10 microg of DNA loaded on macrocarriers clearly yielded a better (up to 56%) result as compared to a single bombardment (30%). Amongst the various plasmids tested, pBI221 gave the highest (100%) GUS positive explants in the leaf callus.     

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     

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.     

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     

Transient expression from microprojectile-mediated DNA transfer in pinus taeda

Summary Transfer of plasmid DNA to Pinus taeda L. (loblolly pine) cotyledon cells by microprojectile bombardment has been demonstrated using beta-glucuronidase (GUS). GUS histochemical staining indicated active enzyme in localized centers (blue spots) 24 hours after bombardment. GUS expression declined during subsequent culture, but remained detectable in meristematic tissue 62 days post-bombardment, however, transgenic shoots were not recovered. Localized GUS expression events resulted predominantly from single-cell events containing one microprojectile. The staining pattern was complex, with indigo found both in the central target cell and in adjacent cells. Cellular damage sustained by GUS-positive cells ranged from undetectable to sufficiently extensive to cause cell death. Microprojectile bombardment provides a useful method to assay transient gene expression in loblolly pine and has potential for the production of transgenic plants in pine.     

Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants

Summary Bacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed β-glucuronidase (GUS). Cells of wildtype Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed.     

Amplification and expression of the β-glucuronidase gene in maize plants by vectors based on maize streak virus

Maize streak virus (MSV) is a geminivirus infecting monocotyledonous plants. Its genome consists of one molecule of circular, single-stranded DNA of 2.7 kb. The viral DNA can be efficiently introduced into maize plants by agroinfection which results in systemic infection. To explore the potential of MSV as a replicative gene vector, a reporter gene coding for β-glucuronidase (GUS) was inserted into the non-coding region of the viral genome. The resulting construct (MSV—GUS) of about 5.9 kb was still able to replicate in cells of maize plants although it was unable to induce viral symptoms. This replication led to a five to 10-fold increase in the mean number of GUS-positive spots per plant as compared with infections with the GUS gene without the MSV replicon. MSV—D—GUS, which differed from MSV—GUS by the deletion of genes V1 and V2 encoding a putative movement protein and the coat protein, respectively, also replicated and produced even more GUS-positive spots. In both MSV—GUS- and MSV—D—GUS-infected plants, the GUS-positive spots were located mainly on the veins of leaves whose primodia had already developed at the time of inoculation and never on the leaves developing later. Thus, neither viral construct was able to move systemically, most probably because the DNAs were too large to be packaged.     

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.     

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.     

Stable transformation via particle bombardment in two different soybean regeneration systems

The Biolistics^® particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for -glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.     

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.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Transient gene expression in vegetative shoot apical meristems of wheat after ballistic microtargeting

A method has been established that allows the targeted delivery of DNA-carrying gold particles to vegetative shoot apical meristems of cereal species. Meristems of 8- to 10-day-old seedlings of wheat ( Triticum aestivum ), rice ( Oryza sativa ) and sorghum ( Sorghum bicolor ) were manually exposed by removal of the coleoptile and the first three to four leaves. Using ballistic microtargeting, equal-sized gold particles of different diameters ranging from 1.0 to 2.0 µm were propelled to meristems by pulses of compressed nitrogen ranging from 9 to 13 MPa. When accelerated by 13 MPa, particles of 1.4 µm or larger penetrated to cells of L2 in 80% of the bombarded wheat meristems. Expression vectors containing either the gene for β-glucuronidase ( gusA ) or genes regulating the anthocyanin biosynthesis ( Bperu and C1 from maize) were delivered to wheat meristems. The level of transient gene expression was dependent on the osmotic pressure of the MS-based medium that was used to mount the explants for shooting: an increase of the sucrose concentration from 2 to 10% in the medium resulted in an increase of transient gene expression from 5 to 25% of the bombarded meristems. Although particles of 1.4 µm were efficiently delivered to L1 and L2, transient gene expression occurred predominantly in the L1 layer. In contrast, up to 10% of the bombarded meristems had expressing cells in L2 when particles of 2.0 µm were used. Ten days after bombardment, GUS-positive sectors in meristems and in leaf primordia were observed. When destructive GUS detection was omitted, between 80 and 90% of the bombarded ex-plants recovered in vitro on basal MS medium and then regenerated to fertile plants in the greenhouse.     

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.     

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     

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.     

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.