Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

• the present invention relates to a method for introducing molecules / particularly genetic material, into intact plant cells.
• introducing molecules such as genetic material, for instance plasmid DNA, RNA, vira or fragments thereof, into animal cells and protoplasts.
• Such known methods include inter alia chemical methods, electroporation and microinjection. It has been shown that such known methods could be implemented in both transient and stable transformation. In general all methods disclosed for obtaining transient expression have turned out to be usable for obtaining stable transformation.
• polyethylene glycol polyethylene glycol
• PEG polyethylene glycol
• polyethylene glycol usually 40% polyethylene glycol 6000, is added to plant protoplasts and genetic material ( rens et al., Nature, 296, 72-74, 1982).
• Polyethylene i ine or poly-L-ornithin can be used correspondingly.
• Electroporation In this case a suspension of animal cells or plant protoplasts is exposed to a short electric pulse of high field strength in the presence of genetic material (Neuman et al., EMBO J., 1, 841-845, 1982; Fromm et al., Nature, 319, 791-793, 1986).
• WO Publication No. 89/02464 discloses the transformation of animal cells with DNA fragments by subjecting the cells to ultrasound treatment sufficient to traumatize the cells without killing them. Due to the similarities between animal cells and plant protoplasts mentioned above it has to be considered obvious that this method can also be used to introduce DNA fragments in plant protoplasts.
• the object of the present invention is to provide a method for introducing molecules, particularly genetic material, into intact plant cells, in an inexpensive and effective way.
• the method according to the invention is a useful alternative to previously known introduction methods.
• the method provides a new approach involving only moderate costs, said method being carried out quickly and simply. Based on introduction experiments already conducted it can be predicted that the method will also turn out to be superior when used for a great number of other biological materials, where known methods are insufficient.
• the plant cells are subjected to an ultrasound treatment considerably milder than the ultrasound treatment used for homogenizing or lysing cells. It is important that the treatment is suitably mild so that a sufficient number of plant cells remain viable.
• the plant cell suspension can advantageously be exposed to ultrasonic waves of a frequency range of from 5 kHz to 10 MHz, particularly from 10 to 100 kHz, and an electric output power (i.e.
• Examples of molecules introducable into plant cells by the method according to the invention include DNA, plasmid DNA, RNA, vira, proteins, lipids, pharmaceutical compositions, small molecules, organelles or fragments of such materials.
• the method according to the invention has advantageously been used to introduce molecules into cells of sugar beet and tobacco plants.
• a particularly suitable medium for plant cells and molecules during ultrasound treatment is CPW comprising from 21 to 28% sucrose.
• the mild ultrasound treatment of the method according to the invention is advantageously carried out using a sound-emitting means having an acute point, said means being only immersed in the upper portion of the medium.
• the method according to the invention is advantageously carried out by using a device comprising a sound-emitting means capable of emitting ultrasound in a medium for a period of up to 10.000 ms.
• a device comprising a sound-emitting means capable of emitting ultrasound in a medium for a period of up to 10.000 ms.
• Such a device is advantageously provided in such a way that it emits ultrasonic waves of a frequency in the range of from 10 to 100 kHz.
• the device according to the invention can be equipped so that the electric power supplied to the sound-emitting means can be adjusted to any given value in the range of from 5 to 300 W and so that the duration of the ultrasou ⁇ treatment can be adjusted within a range of from 100 to 10.000 ms.
• the sound source of the device is advantageously formed in such a way that said device can be immersed in a medium in a suitable vessel, for instance an Eppendorf tube.
• a suitable vessel for instance an Eppendorf tube.
• the present invention has been developed using a device for lysing cells, said device being commercially available from Branson, Eagle Road, Danbury, Connecticut, USA, under the name of Sonifier B 15.
• the device can emit ultrasound of a frequency of 20 kHz.
• the electric power values expressed in watts mentioned in the present specification with claims are electric output powers as read from the output control of the electric power supply unit.
• the ultrasound-emitting means was immersed for approx. 2 to 3 mm measured from the surface.
• Preliminary experiments with a calorimeter have shown that the ultrasonic power imparted to the liquid during experimental procedure is approx. 5 to 10% of the given electric output power.
• the genetic material in question is especially DNA or fragments thereof, such as plasmid DNA.
• the method is also suitable for introducing RNA or fragments thereof as well as vira, for instance for pathological tests.
• the method according to the invention is probably also suitable for introducing proteins, lipids, pharmaceutical compositions, small molecules as well as organelles and virus particles into cells.
• the medium can be any suitable, conventional medium for cells or protoplasts.
• the method according to the invention uses a technique presumbably resulting in a temporary moderate weakening of both the cell membrane and the cell wall, which is shown in intact plant cells in the following examples. The examples thus show that plasmid DNA can penetrate plant cells having been treated with ultrasound.
• a cell suspension culture of sugar beets (Beta vulgaris L.) of the genotype Ml (available from DANISCO A/S, Copenhagen, Denmark) is prepared from callus obtained from embryos.
• the cell suspension is cultured in darkness at 25°C on a rotary shaking table.
• the cells are maintained by sub-culturing in a medium according to Murashige and Skoog (Physiol. Plant., 15, 473-497, 1962) to which 5.7 ⁇ M indol acetic acid and 4.4 ⁇ M benzyladenine was added.
• CPW is an aqueous solution of a mixture of inorganic salts comprising i.a. approx. 10 mM Ca , described by Frearson et al., (Dev. Biol., 33, 130-137, 1973).
• a cell suspension for ultrasound treatment is prepared by removing cells 3 to 4 days after sub-culturing and washing them twice with CPW 13S (i.e. CPW containing 13% sorbitol), finally suspending said cells in CPW 13S at a ratio of 1 part by volume cells to 4 parts by volume CPW 13S. Then plasmid DNA is added to the suspension in 0.35 ml CPW containing 21% sucrose and plant cells (500.000/ml) in an Eppendorf tube, the final plasmid concentration being 45 ⁇ g/ml.
• CPW 13S i.e. CPW containing 13% sorbitol
• the plasmid DNA used is a plasmid coding for the marker enzyme chloramphenicol-acetyltransferase (CAT) , in this case the plasmid pCaMVCN having the code 27-4909, available from Pharmacia LKB Biotechnology, Uppsala, Sweden.
• CAT chloramphenicol-acetyltransferase
• the cells treated by ultrasound are then transferred to petri dishes containing a MS-medium (Murashige and Skoog, cf. above) and are incubated for 2 days at 23°C.
• the presence of CAT-activity is shown by adding ⁇ C-marked chloramphenicol to an extract obtained from the treated cells, whereupon the sample is heated to 60°C for 6 min. After cooling acetyl-coenzyme A is added to the sample, the final concentration of acetyl-coenzyme A being 0.71 mM.
• the introduction of plasmids is assessed by measuring the percentage transformation of chloramphenicol (CA).
• the method used is a modification of the method described by Gorman et al. (Mol. Cell. Biol., 2, 1044-1051, 1982).
• the present example illustrates the introduction of plasmid DNA into intact sugar beet cells of the genotype Ml.
• the suspension culture of sugar beet cells is maintained by sub-culturing as described above and is treated with ultrasound, cultured and analysed in the manner described.
• the results of the measured CAT-activity appear from Table 1.
• the present example illustrates the introduction of plasmid DNA into intact tobacco cells.
• a suspension culture of tobacco cells is maintained by sub-culturing in the manner described above for sub-culturing of cell suspensions with sugar beets, the culture medium, however, being a medium according to Murashige and Skoog (Physiol. Plant. 15, 473-497, 1962), to which were added 0.2 mg/1 2,4-dichlorophenoxy acetic acid, 0.1 mg/1 kinetin, 0.9 mg/1 thiamin hydrochloride and 0.2 g/1 H2PO4, pH 6.0.
• the cells are removed 3 to 4 days after sub-culturing and washed twice with CPW 13S (i.e. CPW containing 13% sorbitol), finally suspending said cells in CPW 13S at a ratio of 1 part by volume cells to 4 parts by volume CPW 13S.
• Samples of 0.35 ml each are taken out and the plasmid pCaMVCN is added to each sample, the final plasmid concentration being 100 ⁇ g/ml.
• the cells are then subjected to ultrasound treatment under the conditions appearing from Table 2. After culturing for 2 days in the above-mentioned medium the cells are extracted and their CAT-activity is measured. The results appear from Table 2.
• the method according to the invention can be used to introduce molecules into intact plant cells.

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• Health & Medical Sciences (AREA)
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• Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Cited By (38)

Citations (3)

• 1989
  • 1989-06-29 DK DK325189A patent/DK168302B1/da not_active IP Right Cessation
• 1990
  • 1990-06-28 WO PCT/DK1990/000166 patent/WO1991000358A1/en not_active Application Discontinuation
  • 1990-06-28 EP EP19900910596 patent/EP0480971A1/en not_active Ceased
  • 1990-06-28 JP JP51026590A patent/JPH05500304A/ja active Pending
  • 1990-06-28 AU AU60432/90A patent/AU645260B2/en not_active Expired - Fee Related

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