Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

Production of transgenic tropical maize with cryIAb and cryIAc genes via microprojectile bombardment of immature embryos

To enhance the level of resistance to insects in tropical maize germplasm we have developed techniques to successfully transform elite tropical maize inbred based on the activity of specific cryI proteins against four major maize pests – corn earworm, fall armyworm, southwestern corn borer and sugarcane borer. Constructs containing cryIAb or cryIAc synthetic genes were used. To generate transgenic plants we have established methods for biolistic bombardment and the selection and regeneration of immature embryos and calli from the elite tropical lines CML72, CML216, CML323, CML327 and hybrids. Transgenic plants resistant to the herbicide Basta^TM contained the bands for the cry, bar and gus genes as detected by Southern blot analyses. A simple leaf bioassay presented varying levels of resistance to Southwestern corn borer of transgenic tropical maize carrying the cryIAc gene. Analyses of the progenies confirmed the sexual transmission of the introduced genes and their stable expression. Received: 25 September 1998 / Accepted: 27 October 1998     

Transformation of white spruce (Picea glauca) somatic embryos by microprojectile bombardment

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of 605 embryos (2.2%) remained GUS positive after two months in culture. Three of those thirteen (23%) embryo-derived tissues consistently showed GUS activity for eight months in culture. These putatively transfomed embryogenic tissues were subjected to Southern blot analysis and the results suggested integration of the gus::nptll gene expression cassette in the white spruce genome.Abbreviations ABA (±)abscisic acid - BA benzyladenine - bp base pair - 2,4-D 2,4-dichlorophenoxyacetic acid - kb kilobase - gus E. coli gene uid A for -glucuronidase - nptll neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid     

Optimizing somatic embryogenesis and particle bombardment of barley (Hordeum vulgare L.) immature embryos

Summary A highly regenerable target tissue and a high-frequency DNA delivery system are required for the routine production of transgenic barley. This project separately optimized tissue culture and particle bombardment parameters. Immature zygotic embryos (0.7 to 1.2 mm) were excised and culture on B5L solid medium. Klages and H930-36 cultivars regenerated significantly more green plants than Sabarlis and Bruce. The regeneration pathway shifted from organogenesis to somatic embryogenesis when maltose was used as the medium carbohydrate source instead of sucrose. More somatic embryos were induced on 5 mg/liter 2,4-dichlorophenoxyacetic acid than 2 mg/liter. Gene delivery was optimized using anthocyanin regulatory genes as a transient marker. A 3-mm rupture disc-to-macrocarrier gap distance, a 1-day prebombardment embryo culture period, and a maltose carbohydrate source were each significantly better than other treatments. Double bombardments per plate, a 6-mm macrocarrier fly distance, and 650-psi rupture discs each had the highest number of transiently expressing cells in individual experiments, although the results were not statistically significant compared to the other treatments. Using the optimized parameters, over 200 cells routinely expressed anthocyanin in a bombarded immature embryo. In tissue culture experiments, 350 to 400 green plants regenerated per 100 immature embryos. The improvement of green plant regeneration and gene delivery forms a strong basis to develop a practical barley transformation system.     

Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment

The green fluorescent protein gene ( gfp ) is a widely used reporter in both animals and plants. Fusions between the plastid rrn promoter or the Escherichia coli trc promoter and the gfp coding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of the arc 6 mutant of Arabidopsis thaliana 2–4 days after bombardment. The fusion of the gfp coding region to the bacterial trc promoter demonstrated that a bacterial promoter is active in chloroplasts in vivo . GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants.     

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.     

Sugars regulate cold-induced gene expression and freezing-tolerance in barley cell cultures

The hypothesis that the extracellular concentration of sugars helps regulate the acclimation of plant cells to cold was tested in this work. Suspension cultures were used to control the concentration of sugars in the medium supplied to barley cell cultures (Hordeum vulgare L. cv. Igri), replacing the medium daily to help maintain the concentration. Freezing tolerance and the levels of mRNA expression of the stress-response genes blt4.9 (coding for a non- specific lipid transfer protein) and dhn1 (coding for a dehydrin) were measured. Similar levels of freezing-tolerance and gene expression were obtained in the experiments as occur during cold-acclimation in the crown of the whole plant. In the cell cultures, cold (6/2 degrees C) did not induce an increase in freezing tolerance or in the expression of detectable levels of blt4.9 or dhn1 mRNAs when only 1 g l-1 sucrose was supplied. However, the cells in this low sucrose medium in the cold were not sugar-starved, indicating that this did not explain the failure of the cells to acclimate when grown in the cold environment. Ten g l-1 sucrose supplied to cells grown in the warm (25 degrees C) induced acclimation to freezing and up-regulation of expression of blt4.9 and dhn1 mRNAs. Osmolality of the medium did not explain this. Thirty g l-1 sucrose induced yet higher levels of freezing tolerance and of blt4.9 and dhn1 mRNAs in cultures grown in either the cold or the warm environment. The results implicate sugars in the regulation of cold acclimation     

Factors affecting transformation of cell cultures from three dicotyledonous pigment-producing species using microprojectile bombardment

Gene transfer methods were established for cell suspension cultures of sweet potato (Ipomoea batatas), ohelo (Vaccinium pahalae) and carrot (Daucus carota, two lines) using micro-projectile bombardment. Several parameters were studied (particle size/type, helium pressure, stage height, DNA concentration, pre-culture period) to determine which significantly affected transformation efficiency. All the physical parameters influenced transient gene expression, with particle size and type having the greatest effect. Cell culture age also affected transformation efficiency in all cell lines. Nuclear DNA conformation (relaxation) as measured by flow cytometry showed no change associated with culture age or transformation efficiency. Agrobacterium-based methods were also tested and in most experiments produced no GUS-expressing loci. Microprojectile bombardment will now be used to study anthocyanin biosynthesis in cell cultures as an alternative source of natural food colourants.     

Application of the chloramphenicol acetyltransferase (CAT) diffusion assay to transgenic plant tissues.

Chloramphenicol acetyltransferase (CAT) activity was quantified in crude extracts from tobacco callus tissues using a modification of a previously reported diffusion assay. We describe here the alterations necessary in applying this rapid and simple assay procedure to plant materials. Due to the high concentration of nonspecific oxidases present in most plant tissues, some type of protective agent is required to maintain enzyme activity. We have tested beta-mercaptoethanol, cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone as protective agents within the initial extraction buffer. We also investigated the effect of heat (60 degrees C, 10 min) and 5 mM EDTA on CAT activity. The highest CAT activity was obtained using 5 mM cysteine plus 5 mM EDTA in 40 mM Tris-HCl (pH 7.8) as the initial extraction buffer followed by a heat treatment. Using this buffer, CAT activity was stable on ice for more than two hours. In our hands, total acetyl-coenzyme A concentration within the assay mixture was found to be saturating at 250 microM and the Km determined to be 100 microM. Assays performed using the same crude plant extract indicate that 1) duplicate assays show less than 1.5% variation in activities and 2) CAT activity increases linearly with respect to volume of extract used.     

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl₂, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.     

Expression of gus in somatic embryo cultures of black spruce after microprojectile bombardment

Immature embryos (stage I) and cotyledonary somatic embryos(stage III) of black spruce [Picea mariana (Mill) B.S.P.] werebombarded with tungsten particles coated with a gene constructcontaining the fusion of gus:: nptll. GUS (ß-glucuronidase)activity was monitored histochemically with X-gluc giving ablue colour where transient gene expression was detected inthe bombarded tissues. A high transient expression of gus wasobserved in stage I embryo cultures 2 d after bombardment (202GUS foci per 300 mg tissue). GUS activity had substantiallydiminished in this material 14 d after bombardment, when grownin liquid LP maintenance medium containing BA (4.4µM),2,4-D (9µM) and 1% sucrose. However, when stage I embryoswere cultured on LP maturation medium containing BA (40 µM),IBA (1 µM), 3.4% sucrose and 0.8% agar, GUS activity after2 d was 335 GUS foci per 300 mg tissue, and the activity wasdetected until 30 d after bombardment. With stage III somaticembryos cultured on LP maintenance medium, 92% showed GUS activity2d after bombardment (16 GUS foci per embryo), and 31 % showedactivity 30 d after bombardment (4 GUS foci per embryo). GUSactivity was still evident in 12% of the embryos (2 GUS fociper embryo) 45 d after bombardment. Key words: Black spruce, gus = E. coli geneuid A encoding ß-glucuronidase, nptll = gene encoding neomycin phos-photransferase, somatic embryos     

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.     

Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin.     

Cloning and expression of a chloramphenicol acetyltransferase gene in cytosine-substituted T4 bacteriophage

We have developed an efficient method for transferring foreign genes into the T4 phage genome. Any foreign genes inserted into the T4 uvsY gene cloned on plasmids can be transferred into a cytosine-substituted T4dC(delta NB5060) phage genome by a replacement type of recombination. To achieve this, we constructed chimeric plasmids which had a chloramphenicol acetyltransferase gene (cat) derived from transposon Tn9 inserted into the Bg/II site within the T4 uvsY gene on pBR322. The cat gene was then transferred by in vivo recombination into the T4dC(delta NB5060) phage genome. Moreover, it was demonstrated that the cat gene in the hybrid T4dC phage was expressed upon phage infection and development.     

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     

Stably transformed herbicide resistant callus of sugarcane via microprojectile bombardment of cell suspension cultures and electroporation of protoplasts

Stably transformed callus of a hybrid sugarcane cultivar (Saccharum species hybrid, CP72-1210) was achieved following high velocity microprojectile bombardment of suspension culture cells, and electroporation of protoplasts. A three-day old cell suspension culture (SC88) was bombarded with gold particles coated with pBARGUS plasmid DNA containing the ß-glucuronidase (GUS) reporter gene and the bar selectable gene that confers resistance to the herbicide basta. The pBARGUS plasmid was also electroporated into the protoplasts of another cell line (SCPP). Colonies resistant to basta were recovered from both sources. Stable integration of the bar gene in the resistant cell lines was confirmed by Southern analysis. In addition, phosphinothricin acetyltransf erase (PAT) activity was also demonstrated in the transformed cell lines.Abbreviations GUS ß-glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - PMSF phenylmethylsulfonyl fluoride - MES 2[N-Morpholino]ethanesulfonic acid - HEPES [N-2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] - PAT Phosphinothricin acetyltransferase - CTAB cetyltrimethylammonium bromide