Advances in alternative DNA delivery techniques

This review describes recent advances in alternative DNA-delivery techniques with particular emphasis on silicon carbide fibers, intact tissue electroporation, electrophoresis and microinjection. The advantages/disadvantages of each method along with a historical overview and theory of practice are discussed.     

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neo^R) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neo^R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo^R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo^R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Inheritance of two foreign genes co-introduced intoPetunia hybrida by direct gene transfer

In previous work, transformedPetunia hybrida plants were obtained by direct gene transfer, using two different genes on separate plasmids (NPT II gene and a cDNA of PEPC from green sorghum leaves). In this study, we have analysed the sexual transmission of the acquired genes by genetic crossing analysis of 2 of the transgenic petunias. The ploïdies of the two clones were determined by flow cytometric analysis showing that one was 2n and the other 4n. Self and back crosses show that the kanamycin character was inherited as a single dominant trait, and that the two clones were heterozygotes for this character. Therefore, the 4n clone probably arises from an endoploidization followed by a transformation event. Southern blot analyses show that all of the resistant progenies which were analysed harboured the kanamycin gene, and expressed the phosphorylation activity in vitro. The DNA of several progenies were also tested for the presence of co-transformed PEPC cDNA sequence. All of the kanamycin-resistant progenies tested contained the second coding sequence, indicating that the two foreign genes might be genetically co-inherited in the transgenic plants. The way in which the two genes are integrated into the genome is discussed.Abbreviations NPT neomycin phosphotransferase - PEG polyethyleneglycol - PEPC phosphoenolpyruvate carboxylase     

Transformation efficiencies and progeny analysis after varying different parameters of direct gene transfer of Nicotiana tabacum protoplasts

Nicotiana tabacum protoplasts were transformed by polyethylene glycol (PEG)-mediated uptake and electroporation, with circular and linear DNA, and with or without X-ray irradiation. We investigated the influence on the transient expression by these parameters as well as on the frequencies for stable transformation. Plants were regenerated and selfed, and the progenies of the transformed plants were analysed and used to compare the pattern of gene integration by these different variations in transformation methods. The results from the transient expression as judged by glucuronidase (GUS) activity, showed electroporation to give higher and more reproducible results than PEG-mediated uptake. Using linear instead of circular DNA increased the rate of stable transformation about 3 times. Including a mild X-ray treatment gave an increase in the same range. When the inheritance of the transferred trait was investigated, it was found that protoplasts transformed with linear DNA resulted in the highest number of plants with single-copy insertions. Protoplasts transformed with circular DNA showed the highest incidence of losing the trait, while plants in which the transformation included an X-ray treatment, had the highest frequency of multicopy insertion events.     

Loss of intracellular glycerol from Dunaliella by electroporation at constant osmotic pressure: subsequent restoration of glycerol content and associated volume changes

The effect of electroporation on Dunaliella tertiolecta at constant osmotic pressure (or water activity) was investigated. The following metabolic and physiological parameters were determined: extracellular and intracellular glycerol content, soluble protein content, photosynthetic oxygen evolution, mitochondrial oxygen uptake, cell volume and cell density. Electroporation conditions are described that released about 10% of intracellular glycerol to the external medium with minimal apparent effects on metabolism. Glycerol release originated from most cells rather than by total rupture of a small proportion of cells. Cell volume, measured on motile cells by video microscopy, reduced by 23% immediately after electroporation. Cell density did not increase. The uptake of mannitol, the major solute in the electroporation medium, was less than 20% of glycerol release. Following electroporation, the intracellular glycerol content and the cell volume both returned to pre-electroporation values after about 30min. Because the cells were maintained at constant external osmotic pressure throughout the procedure, it is concluded that the regulatory mechanism responsible for setting the intracellular glycerol content does not sense external osmotic pressure per se. These findings are consistent with a mechanism that senses a parameter linked directly to cell volume to set the intracellular glycerol content.     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.