Paul F. Lurquin

Gene transfer by electroporation

Electroporation of cells in the presence of DNA is widely used for the introduction of transgenes either stably or transiently into bacterial, fungal, animal, and plant cells. A review of the literature shows that electroporation parameters are often reported in an incomplete or incorrect manner, forcing researchers to rely too much on a purely empirical trial and error approach. The goal of this article is to provide the reader with an understanding of electrical circuits used in electroporation experiments as well as physical and biological aspects of the electroporation process itself. Further, a simple paradigm is provided which unites all electroporation parameters. This article should be particularly useful to those new to the technique.

Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos.

SummaryCowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing β-glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and LipofectinTM increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.

Electroporation-mediated gene transfer into intact nodal meristems in Planta: generating transgenic plants without in vitro tissue culture

Transient expression and stable integration and expression of transgenes were observed in the tissues and offspring of certain leguminous plants after electroporation of DNA into intact nodal meristems in planta. The method described in this article thus allows the study of transgene expression in tissues differentiating from meristematic cells present in the treated buds. In addition, transgenic plants can be recovered in the offspring of electroporated individuals. Therefore, this technique allows the production of transgenic leguminous plants without the need for in vitro tissue culture, often a major hurdle with this family.Transient expression and stable integration and expression of transgenes were observed in the tissues and offspring of certain leguminous plants after electroporation of DNA into intact nodal meristemsin planta. The method described in this article thus allows the study of transgene expression in tissues differentiating from meristematic cells present in the treated buds. In addition, transgenic plants can be recovered in the offspring of electroporated individuals. Therefore, this technique allows the production of transgenic legumious plants without the need for in vitro tissue culture, often a major hurdle with this family.

Gene transfer by cocultivation of mature embryos with Agrobacterium tumefaciens : application to cowpea (Vigna unguiculata walp)

Summary Our studies have shown for the first time that chimeric transgenic cowpea plants could be produced from mature embryos cocultivated with Agrobacterium tumefaciens . Pilot studies demonstrated the possibility of obtaining transgenic cowpea callus cells following cocultivation of excised mature embryos with oncogenic hypervirulent Agrobacterium tumefaciens A281 (pTiBo542/pGA472). Subsequently, cowpea plants chimeric for a gus transgene were obtained by cocultivating mature embryos excised from ungerminated seeds with disarmed A. tumefaciens C58 (pGV2260/p35SGUSINT) carrying a plant-expressable gus gene interrupted by an intron. Studies of reporter transgene expression showed that transformed cells were mostly located in the subepidermal regions of the plant stems.

Coat protein-mediated resistance to pea enation mosaic virus in transgenic Pisum sativum L

Pea (Pisum sativum L.) plants were transformed in planta by injection/electroporation of axillary meristems with a chimeric pea enation mosaic virus (PEMV) coat protein gene contruct. R1 progenies of these plants were shown to harbor the transgene by polymerase chain reaction (PCR) and genomic Southern analysis, while transgene expression was demonstrated by western blot analysis. Transgenic R2, R3 and R4 plants displayed delayed or transient PEMV multiplication and attenuated symptoms as compated to control inoculated individuals.

Stable transformation of eggplant (Solanum melongena L.) by cocultivation of tissues withAgrobacterium tumefaciens carrying a binary plasmid vector.

Stable transformation of eggplant to kanamycin resistance was obtained by cocultivation of cotyledonary and young leaves with the hypervirulent, fully oncogenicAgrobacterium tumefaciens strain A281 carrying plasmid pGA472. No transformation was observed when using the disarmedA. tumefaciens LBA4404 strain carrying pGA472 or when using either strain for cocultivation with eggplant suspension cells.The NPTII enzyme and DNA dot blot assays performed on callus cells growing in the presence of kanamycin indicated both the presence and expression of the foreign gene. The highest proportion of transformed explants was obtained from intact cotyledonary leaf pieces while the highest NPTII enzyme specific activity was detected in callus cells originating from superficially wounded cotyledonary leaf pieces. Kanamycin-resistant plantlets were regenerated after six months in culture.

Biological activity of ethidium bromide - transfer RNA complexes.

It was now established that polycyclic drugs such as acridines or ethidium bromide (EB) are able to bind to polynucleotides other than DNA [l-4] . Their inhibitory action could therefore occur at levels other than the transcription of DNA. The interaction of crude E. coli tRNA and EB has been studied by Bittman [5] who suggested that several kinds of tRNA-dye complexes could exist because of the multispecies nature of tRNA. In a previous communication [6] we showed that proflavine considerably inhibits the loading of leucine on tRNA in a system containing E. coli tRNA and activation enzymes whereas there was no such effect on the loading of leucine in a system containing reticulocyte tRNA and activation enzymes. The inhibition by proflavine of the cross-reaction between E. coli tRNA and reticulocyte activation enzymes strongly suggested that the formation of a proflavine-tRNA complex could account for the observed effect. On the other hand, it was also reported [7] that proflavine did not alter at the same levels the maximal loadings of leucine and valine on tRNA in E. coli systems, suggesting also some specificity of the dye. In this paper, we describe the formation of complexes between EB and purified tRNA,,s and tRNA,s from E. coZi. The latter were found to bind less drug by the presumed intercalation process [8] than unfractionated tRNA and tRNA,,s. Moreover, the extent of formation of valyl-tRNA in a reaction mixture was not affected by EB concentrations which yield respectively 50% and 30% inhibition when the loading on tRNA of leucine and of a mixture of 15

Integration versus degradation of exogenous DNA in plants: an open question.

Publisher Summary This chapter aims to evaluate the relatively numerous and conflicting reports concerning the fate of exogenous DNA applied to plant cells. It reviews that classical plant-breeding techniques have been quite successful, although they suffer from the disadvantage of being slow. However, the advent of somatic plant cell genetics coupled with protoplast fusion, yielding in some cases complementation between mutant alleles, may represent a breakthrough in plant breeding, as it bypasses the laborious procedure of field or greenhouse crosses and allows a direct selection of characters under controlled conditions. Although DNA uptake by plants was reported well before the emergence of these still delicate new breeding techniques, the importance is much affected by the success of the other. If exogenous DNA can be taken up by plants and be phenotypically expressed and stably inherited, the consequences for plant breeding would be far more revolutionary than those of somatic cell genetics alone. The chapter also reviews that in the case of plants, essentially two lines of research are followed in tracing the fate of exogenous DNA: (1) a biochemical approach in which donor DNA is detected by some property such as an isotopic label or buoyant density in CsCl density gradients; and (2) biological experiments in which the host plants are examined for the appearance of novel proteins or functions such as growth under nonpermissive conditions.

Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides

A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress. The minicells so prepared are metabolically active as indicated by the in vivo incorporation of [35S]methionine into plasmid-coded polypeptides.

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacteriumtumefaciens

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype.