WO1998010062A1 - Systeme d'expression genique de graines monocotyledones - Google Patents

Systeme d'expression genique de graines monocotyledones Download PDF

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Publication number
WO1998010062A1
WO1998010062A1 PCT/US1997/015340 US9715340W WO9810062A1 WO 1998010062 A1 WO1998010062 A1 WO 1998010062A1 US 9715340 W US9715340 W US 9715340W WO 9810062 A1 WO9810062 A1 WO 9810062A1
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Prior art keywords
gene
plant
seed
monocot
maize
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PCT/US1997/015340
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English (en)
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Gurdip Brar
David R. Russell
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Monsanto Company
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Priority to AU41734/97A priority Critical patent/AU4173497A/en
Publication of WO1998010062A1 publication Critical patent/WO1998010062A1/fr

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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins

Definitions

  • the field of the present invention is plant transformation systems.
  • the field of the present invention is a system for expressing foreign genes in maize kernels by use of the rice glutelin-1 promoter.
  • Plant Transformation One general object of modern biotechnology is to -genetically engineer crop plants by introducing new genetic traits into the genome of elite plant lines. Plants with new traits, such as insect resistance or herbicide resistance, and artificial manipulations of the agronomic qualities of the crop product are possible once recornbinant genes are introduced into plant lines.
  • the first widely used plant genetic engineering technique was based on the natural ability of the soil- dwelling microorganism Aqrobacterium tumefaciens to introduce a portion of its DNA into a plant cell as a part of the normal pathogenic process. If a foreign gene is inserted into the bacteria in certain ways, the Aqrobacterium can be used to transfer the foreign gene into a plant.
  • Aqrobacterium transformation techniques have been developed for a number of plants, mostly dicotyledonous, but the usefulness of the technique has -2 - varied from plant species to species.
  • Agrobacterium- based transformation systems are limited because they require cell or tissue culture and plant regeneration techniques. Plants lines vary in their amenability to tissue culture and regenerations methods.
  • a maize genetic engineering system which obviates tissue culture and regeneration would represent a significant advantage.
  • One new developing technique for creating transformed plants includes bombarding a cell with accelerated particles carrying genetic information. The first indication of the utility of this technique was a demonstration that DNA constructs could be coated onto tungsten particles and accelerated into onion skin where the genes were transiently expressed, as is described in the specification of U.S. Patent No. 4,945,050.
  • a problem in the development of an accelerated particle transformation process to create transgenic plants is the difficulty of obtaining a germline plant transformation.
  • germline transformation we mean that the germ cells of the plant are transformed in such a way that the progeny of the plant inherit the inserted foreign genetic construct. I "some species, plant genetic transformation has been achieved by the accelerated particle method.
  • European Patent Application No. 301,749 discloses the germline transformation of soybean plants and plant lines. The method disclosed in that published patent application is based on accelerating DNA-coated particles into the excised embryonic axes of soybean seeds. If the bornbarded soybean embryonic axes are treated with high ⁇ ytokinin media, shoots are induced from the treated embryonic axes. When the shoots are cultivated into whole soybean plants, a significant percentage of the plants will have transformed ger lines. 62
  • Accelerated particle transformation has also been successful in treatment of maize suspension cultures. Fromm, gt _!• Bio/Technology 8:833-839 (1990), discloses the creation of transformed maize plants from suspension cultures and from calli created from suspension cultures. Klein, e_£. _ . , Proc. Natl. Acad. Sci . USA 85:4305-4309 (1988), discloses accelerated particle transformation of maize suspension culture cells. Spencer, et aJL. , Theor . Appl . Genet . 79:625-631 (1990) , discloses stable transformed maize callus recovered from suspension culture cells bombarded with DNA-coated accelerated particles. Gordon-Kamm, e_t al.
  • Maize embryos have been bombarded and transient " expression achieved by DNA-coated particles.
  • Klein, e_t al. , Bio/Technology 6:559-563 (1988) discloses particle bombardment of surface cells of an excised maize embryo. This reference does not disclose regeneration from the bombarded embryo, nor inheritance in progeny plants.
  • the cells were bombarded with DNA encoding the beta-glucuronidase (GUS) gene. Because it turns a substrate, 5-bromo-4-chloro-3-indolyl glucuronidide, blue in an in situ tissue assay, beta- glucuronidide, can be histochemically detected. After bombardment and treatment with the GUS substrate, blue spots appeared on the embryo, indicating transiently transformed cells.
  • GUS beta-glucuronidase
  • the 3-phaseolin protein was successfully synthesized, processed and accumulated in rice endosperm. What is needed in the art of molecular biology is a maize kernel gene expression system by which gene products can be highly expressed in a maize kernel. Maize kernels containing high levels of specific heterologous gene products will be very useful for modifying seed composition, altering protein
  • the present invention is a monocot seed, plant, or cell containing a gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene, wherein the promoter and the gene are not natively connected.
  • the gene is a mammalian gene and, preferably, the seed, plant or cell is a maize seed, plant or cell.
  • the product encoded by the foreign gene is expressed at a level of at least 1% of the total soluble endosperm protein. More preferably, the product is expressed at a level of at least 2.5% of the total soluble endosperm 62
  • the product is at least 5% of .'the total soluble endosperm protein.
  • the present invention is also a gene construct comprising the rice glutelin-l promoter operably connected to a gene encoding a non-plant protein, preferably a protein such as an antibody.
  • the rice glutelin-1 promoter is the 4888 bp fragment described in SEQ ID NO:l.
  • the rice glutelin-l promoter is a 1.8 kb fragment of the 4888 bp promoter. This 1867 bp fragment extends from residue 3021 to residue 4888 of SEQ ID NO:l.
  • the present invention is method of creating a transformed monocot plant, seed, or cell. This method comprises the steps of creating the gene construct described above, creating a transgenic plant comprising the gene construct, and allowing expression of the non- plant gene.
  • the rice glutelin-1 promoter operably connected with a foreign non-plant gene provides an expression system in maize kernel that results in the foreign gene expression product comprising a significant amount of the total soluble protein. This abundance of foreign protein will facilitate purification of the product and may also improve the nutritional quality of seed protein. It is a further advantage of the present invention that the foreign gene product may be an antibody protein, thus allowing one to more easily produce or isolate recombinant antibodies.
  • a gene product of agricultural importance may be expressed in the maize kernel.
  • a gene product of agricultural importance may be expressed in the maize kernel.
  • Fig. 1 is an exploded schematic view of a particle acceleration device useful in the present invention.
  • Fig. 2 is a top plan view of the device of Fig. 1.
  • Fig. 3 is an enlarged drawing of the corn immature embryo stage 1.
  • Fig. 4 is a schematic diagram of plasmid WRG5084.
  • Fig. 5 is a schematic diagram of plasmid WRG5086.
  • Fig. 6 is a schematic diagram of plasmid WRG456 .
  • Fig. 7 is a schematic diagram of plasmid WRG5236.
  • Fig. 8 is a schematic diagram of plasmid WRG5243.
  • Fig. 9 is a schematic diagram of plasmid WRG5245.
  • This invention relates to the economical production of heterologous proteins in monocotyledonous (monocot) plants such as maize.
  • the heterologous proteins are pharmaceutical proteins such as antibodies.”
  • the proteins are preferably expressed in monocot seeds such as maize kernels. More specifically, the proteins are preferably expressed in the maize seed endosperm. High levels of expression of heterologous proteins, especially of monoclonal antibodies, have been difficult to achieve in monocots before, partly because of the lack of suitable seed- specific promoters .
  • the present invention is a monocot seed, such as a maize kernel, containing a gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene.
  • the gene product encoded by the foreign gene is preferably 62
  • NeoRx451 a second monoclonal antibody
  • the present invention requires a gene construct in which the rice glutelin-1 promoter is operably connected to a foreign gene.
  • the Examples below disclose one particularly useful embodiment of the rice glutelin-1 promoter that was obtained from Dr. N. Murai (Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, Louisiana 70803-1720) as part of clone pGt5.1 (Bglll) .
  • Dr. N. Murai Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, Louisiana 70803-1720
  • a Hindlll site was inserted into the Bglll site destroying the Bglll site and resulting in pWRG4563.
  • the rice glutelin-1 promoter was removed from pWRG4563 as an approximately 4.9 kb.
  • Fig. 6 is a map of pWRG4564.
  • the rice glutelin-1 promoter was then moved as a Xhol-Hindlll fragment into both pWRG2800 and pWRG2801 to make pWRG4565 (glutelin promoter and leader-tobacco extensin leader and signal peptide-BR96 heavy chain) and pWRG4566 (Glutelin promoter and leader-tobacco extensin leader and signal peptide-BR96 light chain respectively) , respectively.
  • the tobacco extensin fragment contains all of the 5' UTR and the first 26 amino acids of extensin coding region (De Loose, e_t ill., Gene 99:95-100, 1991). 35S-hygromycin resistance cassettes were inserted into both of these plasmids 2
  • the nucleotide sequence of the Kpnl-Hindlll fragment from pWRG4563 was determined and is listed at SEQ ID NO:l. Sequencing of both strands was performed by Retrogen Inc. using Applied Biosystems automated sequencer. The transcription initiation site as described by Okita, e_t al., is at residue 4861 of SEQ ID ( N0:1. Nucleotides -4861 to +27 of the rice glutelin-1 gene (as numbered by Zheng, et al. The Plant Journal 4:357-366 (1993)) correspond to nucleotides 1- 4888 in SEQ ID N0:1.
  • a 1867 bp fragment of the 4888 bp promoter has been demonstrated to promote gene expression, although with less strength than the larger fragment. This 1867 bp fragment extends from the EcoRI site to the Bglll site of pGT5.1 as described in Zheng, gt ⁇ i , The Plant Journal 4:357-366 (1993). This fragment corresponds to residues 3021 to 4888 of SEQ ID NO: l. Therefore, a suitable glutelin-1 promoter will contain at least this 1867 bp sequence. Expression levels may be further increased by adding some intron sequences .
  • a preferable gene construct will comprise at least a rice glutelin-1 promoter operably connected to a foreign non-plant gene.
  • signal peptide sequences are useful for protein products that are expected to be secreted through membranes.
  • the antibodies described below in the Examples were connected to the tobacco extensin signal peptide for proper membrane processing.
  • a portion of the rice glutelin untranslated region sequence (UTR) is a preferable addition.
  • such a plant expression vector system includes the co r ding sequence for the desired foreign gene and appropriate regulatory sequences .
  • the appropriate regulatory sequences might include a translational terminator and signal or leader sequences .
  • a translation or transcriptibnal enhancer may be incorporated between the promoter and the coding region of the genetic sequence.
  • the transforming nucleic acid construct can include a marker gene which can provide selection or screening capability in the treated plant tissues .
  • Selectable markers are generally preferred for plant transformation events, but are not available for all plant species.
  • a selectable marker encodes for a trait in the transformed plant cells which can be selected for by the exposure of the plant tissues to a selection agent.
  • Suitable selectable markers can be antibiotic or herbicide resistant genes which, when inserted in some cells of a plant in culture, would imbue those particular cells with the ability to withstand exposure to the antibiotic or the herbicide.
  • At least one antibiotic selectable marker system can be made to work in maize.
  • a gene coding for resistance to the antibiotic hygromycin from E. coli has previously been found to be useful as a selectable marker in tobacco transformation. Waldron, e_t a_L . , Plant Mol. Biol . 5:103-108 (1985).
  • Such selectable markers do not reliably transfer from species to species, particularly when used on callus or differentiated tissue as opposed to protoplasts or suspension culture.
  • marker gene is one that can be screened by histochemical or biochemical assay, even though the gene cannot be selected for.
  • a suitable 62 is one that can be screened by histochemical or biochemical assay, even though the gene cannot be selected for.
  • GUS gene -11 - marker gene found useful in such plant transformation experience is the GUS gene, discussed above.
  • the use of a GUS gene provides a convenient colorimetric assay for the expression of introduced DNA in plant tissues by histochemical analysis of the plant tissues.
  • the gene of interest sought to be expressed in the plant can be coupled in tandem With the GUS gene. Then the tandem construct can be transformed into plant tissues and the resulting plant tissues can be analyzed for expression of the GUS enzyme. Tissues and plants expressing the GUS enzyme will also be found to be expressing the gene of interest.
  • the present invention is a monocot seed, such as a maize kernel, containing a gene construct comprising the rice glutelin-1 promoter operably connected to a foreign non-plant gene.
  • foreign non-plant gene we mean a gene that is not natively connected to the rice glutelin-l promoter and is not isolated from plant sources.
  • the gene is of mammalian origins or is synthetically created.
  • a “foreign non-plant gene” may also be a synthetic gene that is created to mimic a non-plant gene.
  • a “mammalian gene” may include a synthetic gene designed to mimic a mammalian protein such as an antibody.
  • a "non-plant gene” or a “mammalian gene” may be a chimeric gene.
  • the foreign gene encodes an antibody or a fragment of an antibody.
  • the Examples below demonstrate the successful production of both the heavy and light chains of monoclonal antibody BR96 and antibody NeoRx 51.
  • -12- monocotyledonous plants Most preferably, the myention involves the creation of a transgemc maize plant, seed or kernel.
  • a preferred method of transformation of maize embryos One of skill in the art will realize that these methods may be adapted to work in other monocotyledonous plants such as rice and wheat .
  • a gene construct including the rice glutelin-1 promoter will be effective at enhanced gene expression in a variety of transformation systems and methods.
  • a preferred method of preparing maize embryos, transforming the embryos and regenerating the plants we list.
  • One of skill in the art of molecular biology would realize that variations of this transformation procedure would also be effective.
  • Maize kernels are sterilized and isolated from conditioned ears. Preferably, the ears from which these kernels are taken have been conditioned at 4°C from one to eight days with two to four days being preferred.
  • the maize embryo which is composed of the scutellum ,and the shoot/root axis, is then excised from the kernel.
  • the scutellum is the cotyledon of the maize embryo. Esau, Anatomy of Seed Plants, John Wiley & Sons, New York, p. 477 (1960) .
  • immature embryos are excised.
  • immature embryos we mean that the embryos are approximately between 0.50 mm and 2.50 mm in length are in the developmental stages of early coleoptilar, stage one or stage two, as defined by Abbe and Stein Am, Jour . Bot . 41:285-293 (1954). The embryos just entering stage one are preferred. Mature embryos may be used in the present invention, but it is more difficult to culture these embryos. Very immature embryos may be used, but these smaller embryos are harder to manipulate.
  • the maize embryos are placed on a medium amenable to immature embryo scutella culturing.
  • the embryonic axis should touch the media, and the scutellum should 62
  • the medium should contain the salts and carbon source (sucrose or other sugars) needed for growth, an auxin or auxin-like substance, and agar or other gelling agent .
  • the culturing medium contained chloramben, an aUxin.
  • Other auxins, such as 2,4-D may also be used.
  • the embryos are incubated in the dark for conditioning. Usually, this takes about four days.
  • the callus that develops on the scutellum is a mixture of organogenic and embryogenic callus. Almost every maize line is susceptible to this type of callus culture.
  • FIG. 3 illustrating a stage 1 embryo modelled after Abbe and Stein, Am. Jour. Bot. 41:287 (1954) .
  • the suspensor end 2 is where the callus proliferates on the lower one-half to three-quarters of the scutellum.
  • the top one-half to one-quarter of the coleoptile end 3 of the scutellum is the part that is removed.
  • a dashed line 4 indicates the usual locus of the art .
  • the removed scutellum portion is discarded. 2
  • the dissected embryos are placed on fresh medium and positioned so that the callus tissue is available to be bombarded by the accelerated particles.
  • this medium now contains an osmoticum such as 0.4 M mannitol, so that the embryogenic cells will be partly plasmolyzed. This osmotic condition helps to preserve cell integrity through bombardment.
  • the ernbryos should remain on this medium one to three hours at room temperature in the dark before they are bombarded.
  • A. Preparation of Nucleic Acid-Coated Particles Multiple copies of the nucleic acid construct are prepared by known molecular biology techniques .
  • the transformation process requires carrier particles of a durable, dense, biologically-inert material .
  • Gold is a suitable material for use as the carrier particle.
  • the carrier particles are of extremely small size, typically in a range of 0.7 to 3 microns, so that they are small in relation to the size of the maize target cells.
  • " microcrystalline gold particles are used as carrier particles.
  • a suitable source of microcrystalline gold particles is Degussa/Metz (Lot #7-29020-0, Gold Beads 0.71 ⁇ ) . This product consists of gold particles of high surface area and amorphous shape and size. We found ⁇ that microcrystalline carrier particles of irregular size achieve a higher transformation efficiency than that achieved by spherical gold particles.
  • the genetic material to be inserted into the cells is coated onto the carrier particles. Both heavy chain and light chain plasmids are coated onto the gold beads simultaneously for co- ransformation.
  • the DNA loading ratio for each plasmid was 2 ⁇ g/mg of gold particles. This can be readily done by drying solutions of DNA or RNA onto the carrier particles themselves. Suitable stabilizers can be added to the mixture to help with 2
  • the apparatus utilized in the present invention must be capable of delivering the nucleic acid-coated particles into plant cells in such a fashion that a suitable number of cells can be transformed. At some frequency, the carrier particles lodge within the maize cells and, through a process we do not understand, the genetic materials leaves the carrier particles and integrates into the DNA of the host maize cells. Many types of mechanical systems can accelerate the carrier particles into plant cells. Possible mechanisms include ballistic explosive acceleration of particles, centrifugal acceleration of particles, electrostatic acceleration of particles, or other analogous systems capable of providing momentum and velocity to small particles. The mechanism we used in the Example is based on the acceleration of particles through an adjustable electric voltage spark discharge device which is capable of accelerating a planar carrier sheet at a target surface. This apparatus will be described further below with reference to Figs. 1 and 2.
  • the particle acceleration apparatus is generally indicated at 10 of Fig. 1.
  • the apparatus consists of the spark discharge chamber 12 into which are inserted two electrodes 14 spaced apart by a distance of approximately one to two millimeters.
  • the spark discharge chamber 12 is a horizontally extended rectangle having two openings, 16 and 18, extending out its upward end.
  • the opening 16 is covered by an access plate 20.
  • the opening 18, located on the side of the rectangle of the spark discharge chamber opposite from the electrode 14, is ultimately intended to be covered by a carrier sheet 22.
  • the electrodes 14 are connected to a suitable adjustable source of electric discharge voltage (not shown) .
  • a suitable source of electric discharge voltage includes a capacitor in the size range of one to two microfarad.
  • the voltage of the charge introduced into the capacitor should be adjustable.
  • An adjustable voltage can be introduced readily into such a capacitor through the use of an autotransformer which can be adjustable between a range of one and fifty thousand volts.
  • a high voltage electric switch is provided so that the capacitor can be discharged safely through the electrodes 14 without harm to the operator.
  • a carrier sheet 22 is placed upon the opening 18 of the spark discharge chamber 12.
  • the carrier sheet 22 is a planar sheet of relatively stiff material which is capable of carrying small, inert carrier particles thereon toward the target surface.
  • the carrier sheet 22 is a small sheet of aluminized, saran-coated mylar.
  • the function of the carrier sheet 22 is to crbnvert an outwardly outstanding force produced by the electrodes to a broadly distributed horizontal force capable of accelerating a large number of carrier particles in parallel with an even force.
  • Other kinds of force othe than electric discharge can be used to propel the carrier sheet 22 upward.
  • the force should be adjustable so that the force of travel of the carrier sheet 22 can be adjusted.
  • a retaining screen 24 is approximately 15 millimeters above the opening 18 and the discharge chamber 12.
  • a target surface 26 is placed above the retaining screen 24 at a distance of between 5 and 25 millimeters.
  • the target surface 26 is any suitable culture surface onto which the material to be transformed can readily be placed.
  • An overturned petri dish can conveniently be used for the transformation of plant tissues.
  • the DNA-coated particles are layered onto the top of the carrier sheet 22.
  • the layering is done so as to distribute a relatively even pattern of carrier particles across the entire top surface of the carrier sheet 22.
  • the coated carrier particles are applied to the carrier sheet at a loading rate of .025 to .050 milligrams of coated carrier particles per square centimeter of carrier sheet .
  • the carrier sheet 22 is placed upon opening 18.
  • An oil or water droplet is used to adhere the carrier sheet 22 in place.
  • the target surface 26, with the living plant material thereon, is placed in position above the retaining screen 24.
  • a small droplet of water, preferably 10 microliters, is placed in the chamber bridging between the ends of the two electrodes 14.
  • the access cover 20 is placed in position on top of the spark discharge chamber 12.
  • the entire apparatus is enclosed in a vacuum chamber and a vacuum is drawn down into the range of approximately 400-500 millimeters of mercury.
  • a vacuum is drawn down into the range of approximately 400-500 millimeters of mercury.
  • a supply of helium is bled into the vacuum chamber.
  • the vacuum chamber contains a relative vacuum compared to the atmosphere and the atmosphere within the vacuum contains helium.
  • the lower density of helium combined with the reduced pressure, lowers the drag on both the carrier sheet 22 and the carrier particles.
  • the accelerated particle transformation process is initiated at this point.
  • the voltage from the capacitors is electrically discharged to the electrodes 14.
  • the voltages used in the present process have been in the range of 9-25 kV.
  • the range of 9-10 kV is preferred.
  • the voltage is discharged through the use 62
  • the impact of the radiating shock wave upon the interior of the discharge chamber 12 is focused towards the carrier sheet 22, which is then propelled upward with great velocity.
  • the upwardly traveling carrier sheet 22 accelerates upward at great force until it contacts the retaining screen 24.
  • the displacement of the remaining atmosphere in the chamber with helium assists in the travel of the carrier sheet 22, since helium provides less drag on the flight of the carrier sheet as well as on the carrier particles themselves.
  • the carrier sheet 22 impacts the retaining screen 24 and is retained.
  • the nucleic acid-coated particles in contrast, fly off of the carrier sheet and travel freely toward the target tissues. The small carrier particles then hit the living tissue on the target surface and proceed into the tissue cells.
  • Plants must be created from the bombarded conditioned IES tissue. At either the cellular or plant level, the plants must be screened or selected to segregate the transformed plants from the nontransformed plants. In most particle-mediated plant transformation procedures done without selection, the nontransformed plants will be the large majority of the recovered plants. If a selection agent like hygromycin is used, the number of plants recovered is smaller but the relative proportions of transformant plants recovered is much higher. The selection regimen may not kill all of the non-transformed embryogenic cells, 62
  • the embryo tissue After bombardment, the embryo tissue is fragile. It is considered advantageous to let the bombarded tissues remain on the culture medium for approximately four hours. If a gene encoding a selection marker has been incorporated into the cells, the regenerating plant must be placed on medium containing the selection agent at some point in the growth process. Many independent transformants have been obtained.
  • the expression level is at least 1% of total soluble protein (TSP) in endosperm. More preferably, the level is at least 2.5% of total soluble endosperm protein. Even more preferably, the level is at least 5% of total soluble endosperm protein, and, most preferably, the level is at least 10% of this total soluble protein in endosperm.
  • TSP total soluble protein
  • Total soluble protein from the endosperm is essentially extracted the same way as the whole seeds except that the endosperms (because they are wet) are ground in pestle and mortar.
  • Extraction Buffer (for 500 mis ) 100 mis 0.5M NaP04 (pH 7 .0) final cone . - 100 mM NaP04 20 mis 0 . 5M EDTA (pH 8 . 0) 20 mM EDTA 10 ml ⁇ 10* Triton X-100 0 .2% Triton X-100
  • the buffer should be filter sterilized or made from sterile stocks.
  • Ground material is poured into a 1.5 ml microcentrifuge tube. Approximately 1.5 ml extraction buffer is added. The mixture is vortexed on high for 5-10 seconds and then soaked at 4°C for at least 2 hours. The extractions are sonicated for 1.5-2 minutes. The tubes are centrifuged in a microcentrifuge set at highest speed for 20 minutes at 4°C to remove cell debris. All of the supernatant is transferred to fresh tube.
  • a set of protein standards is prepared using l ug/ul human IgG and unknowns using Coomassie Plus Protein Assay kit from Pierce. Absorbance is read at 595 nM. Unknowns are compared to IgG standard curve to determine total protein in seed extract .
  • TSP total soluble protein
  • Wells are washed 3X with PBS + 0.25% Tween20 (PBST) , using plate washer.
  • Blocking Solution 0.5 g Carnation Milk Powder in 25 mis PBST
  • the plate is incubated at RT for 1 hour.
  • Wells are washed 3X with PBST using plate washer. 62
  • the dilution plate is prepared as follows: load samples into head wells (vertical row) , make 3 or more serial 1:2 dilutions (in PBS) across plate using the 8-channel pipettor. Start with 200 ul (total volume) in head well and 100 ul in each dilution well. Pipet 100 ul from head well into first dilution well and mix. Remove 100 ul from first dilution well and mix with the next well, etc.
  • detection antibody goat anti-human, kappa-specific--HRP conjugate
  • 50 ul is loaded per well and incubated at 37°C for 1 hour.
  • Wells are washed 3X with PBST using plate
  • Substrate solution is prepared according to BioRad kit directions (9 mis TMB peroxidase solution A + 1 ml solution B) , loaded 100 ul per well and incubated at RT for exactly 10 "minutes. The reaction is stopped by loading 100 ul of 1M H 2 S0 4 per well. Absorbance is read at 450 nM.
  • promoters were operably connected to the BR96 heavy chain and light chain genes.
  • the heavy chain and light chain were on two different plasmids.
  • the heavy chain and light chain genes were on the same plasmid.
  • Monoclonal antibody BR96 binds selectively to carcinomas of the colon, breast, ovary and lung; This monoclonal antibody is described in Hellstrom, e_£ al. , Cancer Research 50:2183-2190, 1990, and Trail, e_fc. _ ⁇ l. , Science 261:212-215, 1993.
  • the gene constructs containing both the light and heavy chain of BR96 and the rice glutelin-1 promoter " are described above and at Figs . 4 and 5.
  • WRG5216 maize glob- 3 10 0.08 0.01 to 0.17 17 _ 157**
  • ELISA values were expressed as percent of total soluble protein. ELISA values of RI endosperms only. Scutella were used to obtain R2 plants. "ELISA assays performed on whole seed protein. ELISA values for whole seed protein were similar to endosperm values in transformants with plasmids WRG5117, WRG5216 and WRG5217.
  • gene constructs containing rice glutelin-1 promoter express the foreign mammalian gene, in this case antibody BR96 heavy chain and light chain, to a level of between .15 to 10.1% of the total soluble protein in endosperms . This level stands in sharp contrast to the other promoters evaluated, which never expressed BR96 gene at a level of more than approximately 1% of the TSP.
  • GT3 another rice glutelin promoter, GT3, was examined in transgenic rice.
  • 850 bp of the GT3 promoter (Leisy, v_J_ al., Plant Mol. Biol . 14:41-50, 1989) was fused to the GUS gene. This construct was used to transform Gulfmont rice. Transformants were negative for GUS expression in rice seed (Cooley, e_ a_L., Theoretical and Applied Genetics. 90:97-104, 1995) .
  • Table 3 tabulates the individual rice glutelin-1 promoter transformants that were summarized in Table 1 above.
  • Table 3 experiments we assayed the expression levels of BR96 as percentage of total soluble protein in endosperms of transgenic R maize kernels. Endosperms were separated from scutella to run the ELISA assays, and scutella were used to grow R x plants. The resulting R 2 seeds from R x plants were assayed to determine the transgene transmission and stability of level of expression. The results are presented in Table 3.
  • Rice Glutelin-1 Promoter Provides High Expression Levels of BR96 in Endosperms and Whole Corn Seeds
  • the RI seed of these transformants was analyzed with the following results:
  • the NeoRx451 monoclonal antibody was expressed as 0.4 to 3.5% of endosperm total soluble protein.
  • mean NeoRx451 ELISA values were 5.0% and ranged from 1.6 to 12.8% of total soluble seed protein.
  • NeoRx 451 For producing the glycosylated version of NeoRx 451, the transforming plasmids containing the NeoRx- light and heavy chains (glycosylated) respectively, WRG5243 and WRG5245, are illustrated in Figs. 7 and 9. Ten independent transformants were produced. The x seed of these transformants was analyzed with the following results: The NeoRx451 monoclonal antibody was expressed as 11.0 to 25.7% of endosperm total soluble protein and 0.3 to 15.0% of whole seed TSP (Table 4) . Mean whole seed ELISA values, for differentenfin, « «whi_, 8/10062
  • Rice Glutelin-1 Promoter Provides High Expression Levels of Glycosylated and Non-Glycosylated Forms Of Monoclonal ⁇ ntibody NeoRx451 in Endosperms and Whole Corn Seeds
  • CTAGGCAGGG CAGGCCATTT AGACGAAGCA GAGCAGCTAG TAAACACTAT GCCTATGGAA 2040 CCTGATGCAG TAGTTTGGGG TGCTATCTTC TTTGCTTGTA GGATGCAAGG TAATATCTCT 2100 CTTGGAGAAA AGGCAGCAAT GAAATTGGTA GAAATTGATC CTAGTGATAG TGGAATCTAT 2160 GTGCTACTGG CTAATATGTA TGCAGAAGCG AACATGAGGA AGAAGGCTGA CAAAGTCAGG 2220 GCTATGATGA GACATTTGGG AGTGGAGAAA GTTCCTGGGT GTAGCTGCAT TGAGTTGAAT 2280 GGTGTGGTTC ATGAATTTAT CGTGAAGGAC AAGTCACATA TGGATAGTCA TGCTATTTAT 2340 GACTGCTTGC ATGAGATCAC CCTACAAATA AAGCATACTG CAGATTTGCT TAGCATTTCT 2400 GCGGCTGGTG CGGTGTAGTG TTCTGT

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Abstract

On décrit une construction génique comprenant le promoteur de la glutéline-1 du riz, relié de manière fonctionnelle à un gène étranger non végétal. On décrit également une graine monocotylédone contenant une construction génique comprenant un promoteur de la glutéline-1 du riz, relié de manière fonctionnelle à un gène étranger non végétal. De préférence, le produit génique est exprimé à un niveau d'au moins 1 % de la protéine totale soluble de l'endosperme, et plus préférablement, il est exprimé à un niveau d'au moins 2,5 % de cette protéine.
PCT/US1997/015340 1996-09-03 1997-09-02 Systeme d'expression genique de graines monocotyledones WO1998010062A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078975A2 (fr) * 1999-06-17 2000-12-28 Danisco A/S Promoteur
EP1088061A2 (fr) * 1998-06-15 2001-04-04 The John Innes Centre Procedes et moyens d'expression de polypeptides de mammiferes dans des plantes monocotyledones
FR2799203A1 (fr) * 1999-10-01 2001-04-06 Biogemma Fr Promoteurs specifiques de l'albumen des graines de vegetaux
WO2001075132A2 (fr) * 2000-04-03 2001-10-11 Monsanto Technology Llc Expression et purification de polypeptides authentiques bioactifs issus de plantes
WO2001083792A2 (fr) * 2000-05-02 2001-11-08 Applied Phytologics, Inc. Facteur de transcription d'une plante et expression genetique amelioree
US6991824B2 (en) 2000-05-02 2006-01-31 Ventria Bioscience Expression of human milk proteins in transgenic plants
WO2006066340A1 (fr) * 2004-12-21 2006-06-29 Grain Biotech Australia Pty Ltd Procede permettant l'expression elevee d'immunoglobuline dans des vegetaux
US7417178B2 (en) 2000-05-02 2008-08-26 Ventria Bioscience Expression of human milk proteins in transgenic plants
FR2913694A1 (fr) * 2007-03-13 2008-09-19 Malaysian Palm Oil Board Elements regulateurs d'expression
US8058512B2 (en) 2001-06-05 2011-11-15 David N. Radin Gene expression and production of TGF-β proteins including bioactive mullerian inhibiting substance from plants

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4956282A (en) * 1985-07-29 1990-09-11 Calgene, Inc. Mammalian peptide expression in plant cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4956282A (en) * 1985-07-29 1990-09-11 Calgene, Inc. Mammalian peptide expression in plant cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PLANT MOLECULAR BIOLOGY, 1989, Vol. 14, LEISY et al., "Expression of a Rice Glutelin Promoter in Transgenic Tobacco", pages 41-50. *
PLANT MOLECULAR BIOLOGY, 1991, Vol. 16, TAKAIWA et al., "Analysis of th 5' Flanking Region Responsible for the Endosperm-Specific Expression of a Rice Glutelin Chimeric Gene in Transgenic Tobacco", pages 49-58. *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1088061A2 (fr) * 1998-06-15 2001-04-04 The John Innes Centre Procedes et moyens d'expression de polypeptides de mammiferes dans des plantes monocotyledones
EP1088061A4 (fr) * 1998-06-15 2005-03-16 John Innes Ct Procedes et moyens d'expression de polypeptides de mammiferes dans des plantes monocotyledones
AU778059B2 (en) * 1999-06-17 2004-11-11 Dupont Nutrition Biosciences Aps Promoter
WO2000078975A3 (fr) * 1999-06-17 2001-05-31 Danisco Promoteur
WO2000078975A2 (fr) * 1999-06-17 2000-12-28 Danisco A/S Promoteur
US7053268B1 (en) 1999-06-17 2006-05-30 Danisco A/S Promoter
FR2799203A1 (fr) * 1999-10-01 2001-04-06 Biogemma Fr Promoteurs specifiques de l'albumen des graines de vegetaux
WO2001025439A1 (fr) 1999-10-01 2001-04-12 Biogemma Promoteurs specifiques de l'albumen des graines de vegetaux
US7071378B1 (en) 1999-10-01 2006-07-04 Biogemma Plant seed endosperm specific promoter
WO2001075132A2 (fr) * 2000-04-03 2001-10-11 Monsanto Technology Llc Expression et purification de polypeptides authentiques bioactifs issus de plantes
WO2001075132A3 (fr) * 2000-04-03 2002-06-06 Monsanto Technology Llc Expression et purification de polypeptides authentiques bioactifs issus de plantes
WO2001083792A3 (fr) * 2000-05-02 2002-07-18 Applied Phytologics Inc Facteur de transcription d'une plante et expression genetique amelioree
US6991824B2 (en) 2000-05-02 2006-01-31 Ventria Bioscience Expression of human milk proteins in transgenic plants
WO2001083792A2 (fr) * 2000-05-02 2001-11-08 Applied Phytologics, Inc. Facteur de transcription d'une plante et expression genetique amelioree
US7417178B2 (en) 2000-05-02 2008-08-26 Ventria Bioscience Expression of human milk proteins in transgenic plants
US7589252B2 (en) 2000-05-02 2009-09-15 Ventria Bioscience Plant transcription factors and enhanced gene expression
US7718851B2 (en) 2000-05-02 2010-05-18 Ventria Bioscience Expression of human milk proteins in transgenic plants
US8058512B2 (en) 2001-06-05 2011-11-15 David N. Radin Gene expression and production of TGF-β proteins including bioactive mullerian inhibiting substance from plants
WO2006066340A1 (fr) * 2004-12-21 2006-06-29 Grain Biotech Australia Pty Ltd Procede permettant l'expression elevee d'immunoglobuline dans des vegetaux
FR2913694A1 (fr) * 2007-03-13 2008-09-19 Malaysian Palm Oil Board Elements regulateurs d'expression

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