Electroporation Of Intact Cells Research Articles

Electroporation of intact cells of Streptomyces parvulus and Streptomyces vinaceus.

Various assays of classical PEG-assisted transformation as well as electrotransformation of Streptomyces parvulus IMET41380 and Streptomyces vinaceus NCIB8852 are described. Contrary to the so far reported assays of electrotransforming Streptomyces strains, electroporation in S. parvulus and S. vinaceus was carried out on intact cells, without any lysozyme treatment. In these two strains, the classical PEG-assisted transformation of protoplasts does not work efficiently (10(3) to 10(4) transformants per micrograms of pIJ702 DNA) and electrotransformation gives 10 to 100 times higher yields (10(5) transformants per micrograms of pIJ702 DNA). The electroporation method described here is not applicable to other Streptomyces strains (S. lividans or S. coelicolor).

Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis

The optimal pH conditions for efficient transformation of protoplasts and intact cells were established in avermectin high-producing mutants, ATCC31780 and L-9. Among all factors tested, protoplast buffer pH was elucidated as the most important factor influencing transformation efficiency. The optimal pH of the protoplast buffer for the regeneration of ATCC31780 was 6.5, and using this condition, 4.5 × 106 transformants per μg of pIJ702 were produced. At pH 6.3, the maximal number of L-9 transformants was 1.6 × 105 per μg of the same plasmid. However, the protoplasting process decreased avermectin productivity to half or one-sixth of ATCC31780 or L-9, respectively. To avoid the productivity loss, electroporation of intact cells without lysozyme treatment was developed for these mutant strains even though this method was approximately 100-fold less efficient. In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.

SOMACLONAL VARIATION IN TRANSGENIC PLANTS

This paper summarises our experience of the molecular and field analysis of somaclonal variation within transgenic plant populations. Transgenic rice, poplar and sugarcane plants were produced with different transformation protocols. These were based on protoplast treatment, Agrobacterium tumefaciens infection, particle acceleration or intact cell electroporation, all having in common dedifferentiated cell culture, followed by plant regeneration and selection. Transgenic Arabidopsis thaliana plants have also been produced by an approach that apparently does not require cell dedifferentiation. This is based on in planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. The presence and activity of the foreign gene in the transgenic genonie has been verified by conventional methods (PCR, Southern. Northern and Western blotting). Genomic changes have been evaluated with PCR-based tools (RFLP. RAPD, AFLP, RAMP and AFRP). The statistical analysis of the polymorphic DNA bands produced by these tools provided the basis for answering some basic questions related to the conditions that favour induction, frequency, inheritance and commercial exploitation of transgenic somaclonal variants.

Electroporation-mediated transient gene expression in intact cells of sweetpotato

Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotatoIpomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transientGUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined onGUS gene expression (number of blue spots). MaximumGUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study ofGUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.

Analysis of the uptake of the fluorescent marker 2′,7′-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) by hydrogenosomes in Trichomonas vaginalis

The Duorescent dye 2′,7′-bis-(2-carboxyethyl)-5(and-6)-carboxyftuorescein (BCECF) has been widely used as an indicator of cytosolic pH. Here we report that BCECF localizes to hydrogenosomes (hydrogengenerating organelles found in several phylogenetically separate groups of anaerobic protists) in Trichomonas vaginalis, where it was observable by Duorescence microscopy. Its cellular location was confirmed by treatment of BCECF-loaded cells with diaminobenzidine and hydrogen peroxide together with UV illumination. This produced an osmiophilic precipitate in the matrix of hydrogenosomes, observable by electron microscopy. Use of a short (7.5 min) loading period, loading on ice, use of concentrations of BCECF (acetoxymethyl ester) down to 10 nM, and inclusion of the anion channel blockers probenicid or sulfmpyrazone, or the K+/H+ ionophore nigericin in the loading buffer all failed to prevent hydrogenosomal accumulation of BCECF. This uptake was best observed when intact cells were loaded with the ester form of BCECF, but could also be seen using free BCECF following either incubation with ruptured cells or electroporation of intact cells. Hydrogenosomal BCECF loading was also obtained with washed cell lysates, without cytoplasm or metabolic substrates. We tested a range of other Duorogenic dyes designed for cytosolic labeling, and found that the calcium indicator fura-2 (acetoxymethyl ester) and the cell viability marker Duorescein diacetate also labeled hydrogenosomes. The results illustrate a novel use for BCECF as a Duorescen marker for hydrogenosomes (the first such marker), but present a warning against the indiscriminate use of Duorogenic ester dyes to measure properties ofthe cytosol in hydrogenosome-containing organisms — the dyes may also be indicating the properties of the hydrogenosome.

Molecular analysis of the genome of transgenic rice (Oryza sativa L.) plants produced via particle bombardment or intact cell electroporation

In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits.

Production of Transgenic Kidney Bean Shoots by Electroporation of Intact Cells

We obtained transformed bean shoots by electroporation of intact bean cells with the plasmid pDPG165 containing bar gene conferring herbicide resistance to plants. Transformed shoots were selected from electroporated callus on herbicide containing media. Data of molecular analysis (PCR and Southern blotting) confirmed the insertion of bar gene in the genome of herbicide resistant shoots. Detailed procedures for obtaining regenerative bean callus, optimization of electroporation of intact cells and transgenic shoots are given.

Electroporation of intact cells of Streptomyces parvulus and Streptomyces vinaceus

Various assays of classical PEG-assisted transformation as well as electrotransformation of Streptomyces parvulus IMET41380 and Streptomyces vinaceus NCIB8852 are described. Contrary to the so far reported assays of electrotransforming Streptomyces strains, electroporation in S. parvulus and S. vinaceus was carried out on intact cells, without any lysozyme treatment. In these two strains, the classical PEG-assisted transformation of protoplasts does not work efficiently (10 3 to 10 4 transformants per μg of pIJ702 DNA) and electrotransformation gives 10 to 100 times higher yields (10 5 transformants per μg of pIJ702 DNA). The electroporation method described here is not applicable to other Streptomyces strains ( S. lividans or S. coelicolor).

Transgenic sugarcane plants resistant to stem borer attack

A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis δ-endotoxin was expressed in transgenic sugarcane plants (Saccharum officinarum L.) under the control of the CaMV 35S promoter. Genetic transformation was accomplished by electroporation of intact cells. The levels of recombinant toxin were established and biological activity tests were performed against neonate sugarcane borer (Diatraea saccharalis F.) larvae. Transgenic sugarcane plants showed significant larvicidal activity despite the low expression of CryIA(b).

Production of transgenic sugarcane (Saccharum officinarum L.) plants by intact cell electroporation

We describe an efficient procedure for genetic transformation of commercial sugarcane varieties POJ 2878 and Ja 60-5. The transformation protocol is based on electroporation of a plasmid conferring GUS activity into cell clusters isolated from embryogenic calli. Six to eight weeks after electroporation, Ja 60-5 plants regenerated from electroporated tissues were tested and confirmed to be transgenic using histochemical glucuronidase and Southern hybridization analysis. Electroporation of intact cells is an efficient and reproducible method for sugarcane transformation and may also be useful for transformation of other plants.

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10(5) transformants/micrograms DNA) was achieved.

Construction and characterization of a phage-plasmid hybrid (phagemid), pCAK1, containing the replicative form of viruslike particle CAK1 isolated from Clostridium acetobutylicum NCIB 6444

A bacteriophage-plasmid hybrid (phagemid) designated pCAK1 was constructed by ligating 5-kbp Escherichia coli plasmid pAK102 (AprEmr) and the 6.6-kbp HaeIII-linearized replicative form of the CAK1 viruslike particle from Clostridium acetobutylicum NCIB 6444. Phagemid pCAK1 (11.6 kbp) replicated via the ColE1 replication origin derived from pAK102 in E. coli. Single-stranded DNA (ssDNA) molecules complexed with protein in a manner which protected ssDNA from nucleases were recovered from the supernatant of E. coli DH11S transformants containing pCAK1 in the absence of cell lysis. This suggests that the viral-strand DNA synthesis replication origin of CAK1 and associated gene expression are functional in E. coli DH11S. The single-stranded form of pCAK1 isolated from E. coli supernatant was transformed into E. coli DH5 alpha' or DH11S by electroporation. Isolation of ampicillin-resistant E. coli transformants following transformation suggests that the complementary-strand DNA synthesis replication origin of CAK1 is also functional in E. coli. The coat proteins associated with ssDNA of pCAK1 demonstrated sensitivity to proteinase K and various solvents (i.e., phenol and chloroform), similar to the results obtained previously with CAK1. Following phagemid construction in E. coli, pCAK1 was transformed into C. acetobutylicum ATCC 824 and C. perfringens 13 by intact cell electroporation. Restriction enzyme analysis of pCAK1 isolated from erythromycin-resistant transformants of both C. acetobutylicum and C. perfringens suggested that it was identical to that present in E. coli transformants.

Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and development of a cloning vector

We constructed a cloning vector for use in the plant pathogenic bacterium Clavibacter michiganense subsp. michiganense. The vector pDM100 consists of a 3.2-kb restriction fragment of the Clavibacter plasmid pCM1 joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon Tn5 and the gentamicin acetyltransferase of Tn1696. Both antibiotic resistance genes are efficiently expressed in C. michiganense subsp. michiganense. Although polyethylene glycol-mediated transfection of spheroplasts with the DNA of the C. michiganense subsp. michiganense-specific bacteriophage CMP1 yielded about 3 x 10(3) transfectants per microgram of DNA, in transformations with plasmid DNA only a very few transformants were obtained. However, the transformation efficiency could be improved by electroporation of intact cells, giving about 2 x 10(3) transformants per microgram of plasmid DNA. Since a transformation procedure and a cloning vector are now available, pathogenicity in C. michiganense subsp. michiganense can now be analyzed genetically.

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.