Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Patent
1998-11-24
2000-11-07
Fox, David T.
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
8003202, 800320, 435468, 435474, 435421, A01H 100, A01H 400, C12N 1582, C12N 1587
Patent
active
061439495
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to the field of gene engineering, more particularly to a method for transferring genes into plant cells.
BACKGROUND ART
Methods for transferring foreign genes into host cells, that is, the transformation technique, are fundamental techniques in the field of gene engineering. Such methods are used to analyze functions of transferred genes, produce recombinant proteins, gene therapy, generate useful recombinant plants, etc.
Previously developed techniques for transforming plant cells include the electroporation method, the Agrobacterium-mediated method, the microprojectile bombardment method, and the polyethylene glycol method. In the electroporation method, a DNA is transferred using an electric pulse. The Agrobacterium-mediated method utilizes infection by a terrestrial bacterium, Agrobacterium, which causes a plant tumor. In the microprojectile bombardment method, microparticles adsorbing a DNA are shot into cells. The polyethylene glycol method comprises binding a DNA to polyethylene glycol that is a fusion agent and transferring the binding product into cells by (presumably) an endocytosis-like process.
However, these methods cannot introduce macromolecular DNA fragments larger than 25 kb into cells (Carol, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9975 (1996)). For example, it was difficult to apply the above-described transformation methods to isolate and clone genes of a map-base using a gene map made utilizing a restriction fragment length polymorphis (RFLP) marker. In order to transfer a long-chain DNA into cells, attempts were made to improve the above-described transformation methods. Recently, a long-chain DNA was successfully transferred into plant cells by the Agrobacterium-mediated method and the microprojectile bombardment method [Carol, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9975-9979, (1996), Joyce, M. et al., Plant Cell Reports 14, 299-304 (1995)].
However, the Agrobacterium-mediated method has problems in that a foreign gene to be transferred is inserted into a limited vector system and this method cannot be applied to some plant species. The microprojectile bombardment method has disadvantages in that the apparatus used is expensive and the transfer efficiency is low.
In contrast, the polyethylene glycol method is highly applicable since this method can be performed simply if using a purified DNA and can be applied to an unlimited variety of plant cells. However, the transfer of a long-chain DNA into cells by this method has never been reported.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide a convenient method for transferring a gene that enables introducing a long-chain DNA into plant cells and applies to a wide variety of plants.
As a result of intensive investigation to achieve the above-described objective, the present inventors have found that a long-chain DNA can be transferred into plant cells by elevating the concentration of the DNA to be reacted with polyethylene glycol, thereby completing the present invention.
The present invention relates to the polyethylene glycol method of transferring a gene into plant cells. More particularly, it relates to the steps of: (a) mixing a suspension of protoplasts, polyethylene glycol, and a long-chain DNA to give a final concentration of the DNA of at least 50 .mu.g/ml; (b) collecting, washing, and culturing the protoplasts in a medium; and (c) selecting transformed cells, the steps of: (a) mixing a suspension of protoplasts, polyethylene glycol, and a long-chain DNA to give a final concentration of the DNA of at least 100 .mu.g/ml; (b) collecting, washing, and culturing the protoplasts in a medium; and (c) selecting transformed cells, the step of collecting, washing, and culturing the protoplasts in a medium, a precipitated DNA is dissolved by allowing it to stand or by gently stirring to suspend the protoplasts, 100 kb or more, protoplasts are derived from a monocotyledonous plant, the genus Gramineae, and
In the present invention, the term "long-
REFERENCES:
Japanese Journal of Breeding, vol. 46, separate vol. 2 (1996) Kenjiro Ozawa et al. "The efficiency of introduction of giant DNA (100 kb) in the traormation of Oryza sativa by the PEG process". p. 132.
Ashworth et al., "Assembly of High-Resolution Bacterial Artificial Chromosome, P1-Derived Artificial Chromosome, and Cosmid Contigs", Analytical Biochemistry 224:564-571, 1995.
Ausubel et al., "Minipreps of Plasmid DNA", Current Protocols in Molecular Biology 1:1.6.1-2.10.16, 1991.
Datta et al., "Genetically Engineered Fertile Indica-Rice Recovered From Protoplasts", Bio/Technology 8:736-740, 1990.
Duncan et al., "The Production of Callus Capable of Plant Regeneration from Immature Embryos of Numerous Zea Mays Genotypes", Planta 165:322-4332, 1985.
Hamilton et al., "Stable Transfer of Intact High Molecular Weight DNA Into Plant Chromosomes", Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996.
Fujimura et al., "Regeneration of Rice Plants from Protoplasts", Plant Tissue Culture Letters 2(2):74-75, 1985.
Lorz et al., "Advances in Tissue Culture and Progress Towards Genetic Transformation of Cereals", Plant Breeding 100:1-25, 1988.
Ozawa et al., "High-frequency Somatic Embryogenesis from Small Suspension-Cultured Clusters of Cells of an Intersfecific Hybrid of Oryza", Plant Cell, Tissue and Organ Culture 46:157-159, 1996.
Rao et al., "Physical, Chemical and Physiological Parameters for Electroporation-Mediated Gene Delivery Into Rice Protoplasts", Transgenic Research 4:361-368, 1995.
Tada et al., "Efficient Gene Introduction into Rice by Electroporation and Analysis of Transgenic Plants: Use of Electroporation Buffer Lacking Chloride Ions", Theor Appl Genet 80:475-480, 1990.
Tsukahara et al., "Characterization of Factors Affecting Plantlet Regeneration from Rice (Oryza sativa L.) Callus", Bot. Mag. Tokyo 105:227-233, 1992.
Van Eck et al., "Stable Transformation of Tomato Cell Cultures After Bombardment with Plasmid and YAC DNA", Plant Cell Reports 14:299-304, 1995 .
Ishige Teruo
Ohkawa Yasunobu
Ozawa Kenjirou
Fox David T.
Grunberg Anne Marie
Japan as represented by Director General of Ministry of Agricult
LandOfFree
Method for transferring gene does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for transferring gene, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for transferring gene will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1643090