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

Definitions

• This invention relates to the fields of molecular biology and genetic engineering. More specifically, the invention provides methods of introducing foreign genetic material (mRNA) into plants.
• mRNA foreign genetic material
• this invention provides a distinct improvement over methods presently employed in the art. Endogenous proteins of plants are beneficially augmented with valuable proteins from foreign sources utilizing the methods of this invention.
• this invention embraces a transgenic plant expressing beneficial exogenous proteins produced by obtaining a sample of mRNA encoding the exogenous protein, incubating and inoculating seed of the plant with the mRNA under conditions whereby the mRNA enters the seed, germinating the seed and growing the transgenic plant from the seed.
• the sample of mRNA is obtained from soybeans, most desirably from soybean cotyledons or sprouts.
• the sample of mRNA is obtained by transcribing an isolated DNA molecule that encodes the protein, such as a DNA molecule encoding a medically useful protein, preferably a human protein) such as ⁇ -interferon, interleukin 1, interleukin 2 and human growth hormone.
• a medically useful protein preferably a human protein
• the plant is corn and most desirably corn strain 27-1 or 85089.
• this invention embraces a transgenic plant, preferably corn, expressing soy globulin protein, human interleukin 1, human interleukin 2, or human ⁇ -interferon.
• the corn is strain 27-1; alternatively the corn may be strain 85089.
• mRNA from soy cotyledon is isolated and purified and used for micro injection of com seed. The treated seeds are then germinated and resulting plants assayed for exogenous protein expression. Following microinjection and transformation, protein presence and content in propagated transgenic com plants is analyzed by protein detection techniques. The presence of soy DNA in the transformed com is confirmed by Southern blotting using soy globulin specific radiolabeled DNA probes.
• mRNA may be isolated from soy sprouts. Additionally, other strains of com may be utilized, including com strain 85089. The mRNA may be microi ⁇ jected into recipient seeds once or twice or even more times. The methods described result in successful production of transgenic com plants expressing soy globulin protein.
• mRNA Messenger Ribonucleic Acid is a temporary complementary copy of the sense strand of protein coding DNA. In eukaryotes, this major intermediate of gene expression is transcribed from protein coding genes by RNA polymerase ⁇ . It is usually transcribed as a relatively long pre-mRNA which is then processed, still within the nucleus. Further post-transcriptional modifications are made to most eukaryotic mRNA's to add a 5' cap structure and a 3' poly A tail.
• Transgenic Plants Plants which have been engineered via recombinant nucleic acid techniques to express exogenous proteins encoded by other plants or animals.
• Southern Hybridization Analysis A technique developed by Edward Southern in 1975 in which denatured DNA is transferred from agarose gels in which fragments have been separated by electrophoresis to a nitrocellulose or a nylon membrane laid over the gel, before hybridization with a complementary nucleic acid probe. This step is required as hybridization to the gel itself is very inefficient.
• the technique is ubiquitous in molecular genetics and its numerous applications usually revolve around the identification of a particular DNA sequence within a mixture of restriction fragments, for example to determine the presence, position, and number of copies of a gene in the genome. It is also an integral technique used in DNA typing.
• Western Blotting A method for detecting one or more specific proteins in a complex protein mixture such as a cell extract, which is named by analogy with Southern hybridization to detect DNA sequences and Northern blotting to detect RNA's.
• the procedure involves fractionating the protein mixture by denaturing SDS- PAGE gel electrophoresis and transferring and immobilizing the mixture onto a solid membrane of either nitrocellulose or nylon by electro-blotting.
• the loaded membrane is then incubated with an antibody raised against the protein of interest.
• the antibody-antigen complex so formed on the membrane can then be detected by a procedure which involves the application of a secondary antibody, raised against the first antibody, and to which an enzyme has been covalently linked.
• the insoluble reaction product generated by enzyme action can then be used to indicate the position of the target protein on the membrane.
• Ouchterlony Double Agar Immunodiffusion Assay An assay in which antigen and antibody are placed in wells cut in an agar gel which then diffuse towards one another and precipitate to form an opaque line in the region where they meet in optimal proportions. A preparation containing several antigens often gives rise to multiple lines. The immunological relationship between two antigens can then be assessed by setting up precipitation reactions in adjacent wells: The lines formed by each antigen may be completely confluent, indicating immunological identity. They may show a spur, as in the case of partially related antigens, or they may cross, indicative of unrelated antigens.
• Methods of the invention entail isolation and purification of mRNA from a selected source. Following isolation, mRNA is micro injected separately into plant seeds (e.g., com kernels), which are thereafter planted and allowed to grow to maturity. Following transformation, protein content in the resulting generations of plants is analyzed. The presence of the foreign protein in the treated plant is demonstrated via immunological and biochemical methods.
• plant seeds e.g., com kernels
• the method of the invention has the following steps, resulting in the production of transgenic com in which the transgene is stably inherited by subsequent generations: 1. Isolate or otherwise obtain the desired mRNA.
• any mRNA for which expression in plants is desired is suitable for use in the present invention.
• mRNA encoding major structural proteins of plants, e.g., soy globulins
• mRNA encoding proteins having useful biological activity e.g., human growth hormone (HGH) and human ⁇ -interferon.
• HGH human growth hormone
• mRNAs that have been introduced into plants by the methods of the invention include the following: into com variety 85079: soy globulin, human growth hormone, human interleukins 1, ⁇ and 2, human ⁇ -interferon; into com variety 27-1: soy globulin; into com variety 340: human interleukin 2; into American sweet com: soy globulin and human ⁇ -interferon; into rice: soy globulin into wheat: soy globulin and (tentatively) chicken myosin.
• mRNAs contemplated for use in the present invention include, but are not limited to, human albumin, human urokinase plasminogen activator, hiruden (anticoagulant from leech) and human thymosin-4.
• the invention contemplates use of any plant or animal mRNA.
• mRNA is isolated according to standard methods, e.g., as disclosed by Sambrook et al., Molecular Cloning. Cold Spring Harbor Laboratory (1989) or Ausubel et al. (eds) Current Protocols in Molecular Biology. John Wiley & Sons (2001).
• a suitable quantity of the mRNA may be obtained by isolating a total mRNA component from a tissue that expresses the protein, e.g., soy cotyledons or sprouts.
• the mRNA may be produced by in vitro transcription of an isolated DNA molecule that encodes the protein, e.g., ⁇ -interferon- encoding DNA from human cells. In vitro transcription systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or BRL, Rockville, Maryland.
• seeds of plants for which genetic modification is desired are obtained.
• These may be seeds of any plant species, without limitation.
• they are seeds of agronomically important crops and are amenable to microinjection techniques.
• they are seeds from com or wheat.
• they are seeds from rice.
• they may be from other agriculturally important plants such as soybean, barley, alfalfa, sorghum and rye, or they may be seeds of vegetable or fruit crops, including but not limited to tomato, carrot, cucumber, pea, broccoli, cauliflower, cabbage, spinach, and the like.
• any variety is suitable. Particularly suitable are varieties 27-1 and 85089.
• Seeds are soaked in water for a suitable time, e.g., 2-3 days for com, such that they are fully imbibed and amenable to microinjection.
• Microinjection is accomplished by standard techniques, through the use of commercially available micro injection systems. In com, the microinjection needle is placed as close to the central axis of the seed as possible.
• a suitable amount of mRNA to be injected into com is 1 ⁇ g per ⁇ l distilled water.
• the seed may be injected more than once before it is planted, and it may be injected with more than one specific mRNA. Following injection, the seeds are planted and grown, and suitable parts of the grown plants are analyzed for the presence of the protein encoded by the injected mRNA.
• Such analyses are well known to those of skill in the art. They range from protein isolation (e.g., actually purifying the foreign protein and subjecting it to amino acid composition or sequencing analysis) or detection techniques (e.g., immunological techniques such as Western blotting, immunopreciptiation or
• EXAMPLE 1 Stable Transformation of Com with mRNA Encoding Soybean Globulin Genetic transformation of plants based on transfer of information encoded by mRNA, rather than DNA, has been demonstrated in com. mRNA was extracted from the cotyledon and sprout of the soybean and both mRNAs were separately introduced into com kernels which were then planted. Materials and Methods Isolation of mRNA: The cotyledon and/or sprouts (shoot and root axis) of the germinating soybean were used for the extraction of mRNA according to published procedures (Niu, M. C, L. C. Niu, C. Ma, Z. P. Lin and Y. L. Zhang (1980) Scientia Sinica, 23: 119-122.). Eighty percent of the poly A mRNA in soy encodes soy globulin protein (1 IS and 7S).
• Com seeds from strains 27-1 and 85089 were treated separately and analyzed as follows: 200 com seeds, i.e. kernels as a batch were washed twice using tap water. They were then soaked in double distilled water at 4NC for 48 to 72 hours. During this time the water was changed 2 or 3 times. The imbibed seeds were then microinjected with soy mRNA, (isolated from either soy sprout or soy cotyledon) using a commercially available micro injector which is a graduated glass tube of 100 microliter capacity with a stainless steel needle attached. The needle was inserted into the seed as close to the central axis as possible. The amount injected was 1 microgram of soy mRNA in 1 microliter of double distilled water. Over 90% of the injected seeds germinated and grew into com plants.
• Protein Extraction from the Com Kernels Groups of six kernels were soaked overnight in double distilled water at 4°C. After removing the outer skin they were dehydrated by acetone soakings, then de- fatted by ether soakings and then ground into a fine powder. Protein was extracted with 3 ml of extraction fluid (10% glycerine, 5% 2-mercaptoethanol, 2.3% SDS, 8 M/L urea, 0.02% of bromophenol blue in 0.625 M/L Tris-HCl, pH 6.8). The sediment was removed by centrifugation (4000 m for 10 minutes.). The supernatant was boiled 3 mins. and centrifuged (5000 ⁇ m for 5 minutes.). The supernatant was then stored at -20°C for future analysis.
• extraction fluid 10% glycerine, 5% 2-mercaptoethanol, 2.3% SDS, 8 M/L urea, 0.02% of bromophenol blue in 0.625 M/L Tris-HCl, pH 6.8.
• DNA isolated from com treated with soy globulin mRNA was subjected to Southern hybridization analysis with 32P-labeled cDNA probes specific for soy globulin RNA. This revealed presence of soy globulin nucleotide sequences in the treated com.
• mRNA used for microinjection was prepared from at least two sources in the soy plant, e.g. the cotyledons and the sprouts.
• Microinjected com seeds were planted and the com kernels of the first generation were harvested.
• SDS-PAGE of isolated com protein from the first generation revealed an additional band at approximately 67 KD.
• Ouchterlony double agar immunodiffusion assays using rabbit anti-soy protein semm demonstrated that the additional band was of soy origin and due to the presence of soy protein globulin.
• the kernels of the first generation were then planted in order to see if the soy protein globulin was transmitted to the next generation.
• Extracts from the kernels of the first generation of com were analyzed for detection of soy protein as follows: SDS-PAGE: The com kernels of one ear of com constituted one sample. A total of 134 samples were analyzed from the mRNA- treated group and 10 from the control. Of the 134 samples in the mRNA-treated group, 29 exhibited an additional band, and the balance did not. An additional band was observed at 67 KD in the treated com extracts. No new band was observed in control extracts isolated from untreated com.
• Soy sprout mRNA was microinjected once or twice into com strain 85089.
• the data indicate that a second injection of soy sprout mRNA into com strain 85089 did not result in an increase in the number of plants exhibiting an additional band, as shown in Table 1 below.
• the Ouchterlony Double Agar Diffusion Test Based on the results from the SDS-PAGE, com kemel-extracted protein with and without the additional band, and from the control kernels, were subjected to the double agar diffusion test with rabbit anti- soy protein serum. All of the com protein with the additional band reacted to the anti- serum as shown by the precipitation line, but varied in intensity. See Table 2, below:
• Extracts from the kernels of the second generation of com were analyzed as 5 follows: The Western Blotting Technique: The kernels of the first generation were planted and harvested as the second generation. The kernel extracts from this second generation were subjected to the Western blotting technique. The results were that two bands appeared between 60-70 KD. No band was observed in the control.
• the demonstration of soy protein in the kernels of the second generation of com illustrates 10 that the soy mRNA transferred genetic information into the com genome, such that soy globulin protein is expressed in the next generation.
• Southern Blotting Finding the soy protein in the second and succeeding generations of com indicates that the mRNA encoded genetic information has been reverse transcribed and inco ⁇ orated into the com genome. This is presumably due to 15 the presence of reverse transcriptase in the com seed. Southern blotting demonstrated presence of soy globulin encoding DNA in the transformed com. The presence of the hybridizing band provides evidence of reverse transcription of the injected soy mRNA. No soy derived nucleic acid sequences were detected in DNA obtained from untreated com. To summarize, identification of soy protein in the com was carried out in three steps. The first method employed was SDS-PAGE. An additional band from the com extract grown from the mRNA-treated kernels was observed.
• This band was absent in extracts isolated from untreated com.
• the extract of kernels exhibiting the additional band were analyzed by an Ouchterlony double agar immunodiffusion test using rabbit anti-soy protein semm. A precipitation line was found between the anti-serum and the extract from the kernels exhibiting an additional band by SDS-PAGE (Table 2). No line appeared between the anti-serum and the control extract.
• the kernels of the first generation were planted again. The com grown from these kernels was harvested and the protein extracts analyzed by Western blotting.

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• 2001
  • 2001-02-27 CN CNA018231772A patent/CN1555218A/zh active Pending
  • 2001-02-27 WO PCT/US2001/006104 patent/WO2002067663A1/en not_active Application Discontinuation
  • 2001-02-27 EP EP01924097A patent/EP1370130A4/de not_active Withdrawn

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