Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ballas N, Zakai N, Loyter A: Transient expression of the plasmid pCaMVCAT in plant protoplasts following transformation with polyethyleneglycol. Exp Cell Res 170: 228–234 (1987).
Bartlett GR: Phosphorus assay in column chromatography. J Biol Chem 234: 466–468 (1959).
Carmon Y, Czosnek H, Nudel U, Shani M, Yaffe D: DNAase I sensitivity of genes expressed during myogenesis. Nucleic Acids Res 10: 3085–3098 (1982).
Deshayes A, Herrera-Estrella L, Caboche M: Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid. EMBO J 4: 2731–2737 (1985).
Fraley RT: Liposome mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts. Plant Mol Biol 2: 5–14 (1983).
Fraley RT, Dellaporta SL, Papahadjopoulos D: Liposome-mediated delivery of tobacco mosaic virus RNA into petunia protoplasts: A sensitive assay for monitoring liposome-protoplast interactions. Proc Natl Acad Sci USA 79: 1859–1863 (1982).
Fromm M, Taylor LP, Walbot V: Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci USA 82: 5824–5828 (1985).
Fukunaga Y, Nagata T, Takebe I: Liposome mediated infection of plant protoplasts with tobacco mosaic virus RNA. Virology 113: 752–760 (1981).
Gad AE, Bental M, Elyashiv G, Weinberg H, Nir S: Promotion and inhibition of vesicle fusion by polylysine. Biochemistry 24: 6277–6282 (1985).
Gad AE, Elyashiv G, Rosenberg N: The induction of large unilamellar vesicle fusion by cationic polypeptides: the effect of mannitol, size, charge density and hydrophobicity of the cationic polypeptides. Biochim Biophys Acta 860: 314–324 (1986).
Gad AE, Silver BL, Eytan GD: Polycations induced fusion of negatively-charged vesicles. Biochim Biophys Acta 690: 124–132 (1982).
Gorman CM, Moffat LF, Howard BH: Recombinant genomes which express chloramphenicol acetyl transferase in mammalian cells. Mol Cell Biol 2: 1044–1051 (1982).
Herrera-Estrella L, Depicker A, VanMontagu M, Schell J: Expression of chimeric genes transferred into plant cells using a Ti plasmid derived vector. Nature (London) 303: 209–213 (1983).
Ito H, Fukuda Y, Murata K, Kimura A: Transformation of intact yeast cells treated with alkali cations. J Bacteriology 153: 163–168 (1983).
Kanchanapoom K, Boss WF: The effect of fluorescent labelling on calcium induced fusion of fusogenic carrot protoplasts. Plant Cell Rep 5: 252–255 (1986).
Kao KN, Michayluk MR: A method for high frequency intergenic fusion of plant protoplasts. Planta 115: 355–367 (1974).
Linsmaier EM, Skoog F: Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18: 100–127 (1965).
Lurquin PF, Kado CI: Escherichia coli plasmid pBR313 insertion into plant protoplasts and into their nuclei. Mol Gen Genet 154: 113–121 (1977).
Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Nagata T, Okada K, Takebe I, Matsui C: Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse phase evaporation vesicles (liposomes). Mol Gen Genet 184: 161–165 (1981).
Nir S, Bentz J, Wilschut J, Duzgunes N: Aggregation and fusion of phospholipid vesicles. Progress in Surface Science 13: 1–124 (1983).
Okada K, Takebe I, Nagata T: Expression and integration of genes introduced into highly synchronized plant protoplasts. Mol Gen Genet 205: 398–403 (1986).
Olson F, Hunt CA, Szoka WJ, Papahadjopoulos D: Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta 557: 9–23 (1974).
Rigby P, Dieckman M, Rhodes C, Berg P: Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J Mol Biol 113: 237–251 (1977).
Rouze P, Deshayes A, Caboche M: Use of liposomes for the transfer of nucleic acids: optimization of the method for tobacco mesophyll protoplasts with tobacco mosaic virus RNA. Plant Sci Lett 31: 55–64 (1983).
Sela I, Reichman M, Weissbach A: Comparison of dot molecular hybridization and enzyme-linked immunosorbent assay for detecting tobacco mosaic virus in plant tissues and protoplasts. Phytopathology 74: 385–389 (1984).
Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517 (1975).
Straubinger RM, Papahadjopoulos D: Liposomes as carriers for intracellular delivery of nucleic acids. Methods in Enzymology 101: 512–527 (1983).
Szoka F, Papahadjopoulos D: Preparation of liposomes with large internal aqueous space and high capture by reversephase evaporation. Proc Natl Acad Sci USA 78: 4194–4198 (1978).
Watanabe Y, Ohno T, Okada Y: Virus multiplication in tobacco protoplasts incubated with tobacco mosaic virus RNA encapsulated in large unilamellar vesicles-liposomes. Virology 120: 487–490 (1982).
Wilschut J, Nir S, Scholma J, Hoekstra D: Kinetics of Ca++ induced aggregation and fusion of cardiolipin/phosphatidylcholine vesicles: bilayer destabilization occurs during vesicles aggregation. Biochemistry 24: 4630–4636 (1985).
Zilberstein A, Koth T, Altshulerss Y, Lers A, Zamir A: Characters of DNAase activity in tobacco leaf extract. Plant Sci 52: 57–65 (1987).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Rosenberg, N., Gad, A.E., Altman, A. et al. Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts. Plant Mol Biol 10, 185–191 (1988). https://doi.org/10.1007/BF00027395
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00027395