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• • C—CHEMISTRY; METALLURGY
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  • 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
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8203—Virus mediated transformation
• • 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
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8205—Agrobacterium mediated transformation
• • 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

Definitions

• RNA virus-based vectors can spread to non-target plants by mechanical means and/or by insects. Such spread can be prevented by using vectors that can replicate and/or move only in target plants expressing the appropriate trans-acting factors.
• Another aspect of the subject invention pertains to a method of genotypically or phenotypically modifying a host cell, comprising introducing a DNA-launching platform which encodes a viral RNA molecule and an exogenous RNA segment in a location which does not disrupt the replication of said viral RNA segment or said exogenous RNA segment, whereby the exogenous RNA segment confers a detectable trait in the host cell.
• the subject invention applies to a wide array of plant cells.
• Figure 7 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
• Figure 14 shows the efficient replication of launched BMV RNA3 in (la + 2a)- transgenic plants.
• Figure 18 shows the successful GFP expression from the launched BMV RNA3 in protoplasts.
• SEQ DD NO. 7 pB12LR8 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
• SEQ DD NO. 8 pB12LR9 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
• the subject invention is directed towards a method of transfection employing a DNA-launching platform which encodes a modified viral RNA molecule comprising an RNA viral component attached to an exogenous RNA component and a DNA-dependent RNA pol promoter.
• the DNA-dependent RNA pol promoter is preferably but not necessarily fused within up to 10 nucleotides of the 5 ' transcriptional start site of the modified viral RNA molecule, and more preferably within up to 5 nucleotides of the 5' transcriptional start site.
• RNA-launching platform comprising the RNA3 viral replication segment, as well as the exogenous RNA of interest
• RNA3 viral replication segment as well as the exogenous RNA of interest
• DNA-launching platforms could be also derived from either RNA1 or RNA2.
• sequence encoding the la protein could be replaced with an exogenous RNA; replication would require the expression of la (e.g., separate expression plasmid).
• the DNA- launching platform also comprises a ribozyme situated proximate to the 3' end of the modified RNA3, wherein said ribozyme cleaves the RNA3 at the 3' end.
• viral segments from other known viruses, and/or subviral agents can be used to formulate DNA-launching platforms of the subject invention.
• BMV is merely one representative example of the many viruses suitable for practicing the subject invention. It is widely accepted that principles on which the subject invention is based are broadly applicable to a myriad of viruses.
• RNA viruses are seed transmitted from one generation to the next. This property can be exploited to effect genotypic transformation of a plant. That is to say, the modified RNA remains transmissible from one generation to the next, just as seed-borne virus infections are transmitted from one generation to the next.
• Binary vectors for expressing the BMV la and 2a proteins in plants were constructed. Starting with the pBI101.2 construct (Clontech, Palo Alto, CA), the GUS gene was removed by first cutting the construct with EcoRI and SnaBI. The overhanging restriction fragment ends were filled in by treatment with Klenow fragments and dNTPs. The restriction fragment ends were religated forming the pB 101.2LR1.
• a DNA-launching platform having a BMV RNA3 with a GUS gene insertion wherein the GUS is downstream of an additional BMV subgenomic promoter was constructed.
• the pB3LRl 5 construct was cut with Aval and the restriction fragment ends were filled in with Klenow fragment and dNTPs. Construct was then cut with Clal and dephosphorylated.
• the pB3MI22 was cut with Clal and Stul and a B3GUS fragment was isolated. The isolated B3GUS fragment was then ligated to the cut pB3LR15 construct to form a new construct of pB3GUSCPLRl 9 ( Figure 5).
• a DNA-launching platform wherein the BMV RNA3 coat protein was replaced with the SHMV (Sunn hemp mosaic virus) coat protein and the GUS gene was inserted downstream of an additional BMV subgenomic promoter was constructed.
• the pB3RS4 (Sacher et al, 1988) was cut with Aval, blunted with Klenow fragment and dNTPs, and cut with Kpnl.
• the SHMV coat protein fragment was isolated using a low-melting agarose gel.
• the pB3GUSLRl 7 was cut with Stul and Kpnl and dephosphorylated.
• the SHMV coat protein fragment was ligated to the cut pB3GUSLRl 7 thereby forming pB3GUSCPLR24 ( Figure 7).
• NTl Plating Medium (1 liter) was made with NTl medium and 72.86 g mannitol, the pH was adjusted to 5.5-5.7, and the resulting mixture was autoclaved.
• the cells were centrifuged at 800 rpm for 5 min. The supernatant was discarded. About 40 ml of wash solution was added, cells were resuspended and were centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were then resuspended in three volumes of enzyme digestion solution, and incubated for 60 min. at room temperature.
• the cells were resuspended in 1 ml of ice-cold 20 mM CaCl 2 solution. 0.1 -ml aliquots were dispensed into prechilled eppendorf tubes. About 1 ⁇ g of plasmid DNA was added to the cells. The cells were frozen in liquid nitrogen. The cells were thawed by incubating the test tube in a 37°C water bath for 5 min. 1 ml of YEP medium was added to the tube and incubated at 28°C for 2-4 h with gentle shaking to allow the bacteria to express the antibiotic resistance genes. The tubes were centrifuged for 30 s and the supernatant solution was discarded. The cells were resuspended in 0.1 ml YEP medium. The cells were plated on a YEP agar plate containing selection antibiotic(s) and incubated at 28 °C. Transformed colonies appeared in 2-3 days.
• Transgenic N. tabacum and N. benthaniana plants were produced according to the procedures discussed above.
• the plants were transfected with a DNA-launching platform containing a GUS gene (Figure 5a) by particle bombardment as described in Examples 5 and 7.
• the plants were incubated for 3-5 days and then assayed for ⁇ -glucuronidase (GUS) activity using 1 mg/ml X-Gluc (5-bromo-4-chloro-3-indolyl glucucuronide) as substrate in 0.1M potassium phosphate buffer, pH 7.0, 50 ⁇ M potassium ferrocyanide, and 2% Triton® X-100.
• GUS ⁇ -glucuronidase
• Example 9 Transfection of Transgenic Plants Expressing BMV la. 2a. 3 a. and CP
• BMV RNA3 derivatives contained the GUS gene in place of the coat protein ORF ( Figure 5a) (these were inoculated with or without coat protein expression plasmid, Figure 5b), or had the BMVCP gene translated from an additional subgenomic RNA driven from BMV or CCMV subgenomic promoter ( Figures 5c and 5d), or had the SHMV coat protein translated from an additional BMV subgenomic RNA ( Figure 7b).
• N. benthamiana plants were transfected using a particle bombardment as described above with a DNA-launching platform for BMV RNA3 having the GFP gene in place of BMV coat protein (Figure 6e).
• the GFP expression was determined 24 hrs post inoculation using a fluorescent microscope.
• Figure 17 shows the successful expression of GFP in (la + 2a)- transgenic N. benthamiana.
• Example 16 Transfection of Progeny From ( 1 a+2a -Transgenic N. benthamiana With BMV R ⁇ A3 DNA-Launching Platform
• Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator.
• Such factors may include the viral replicase (126K/183K), movement protein (30K), or coat protein (17.6K).
• At least one cis- acting sequence necessary for TMV RNA replication is removed from transgenes.
• the transacting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
• PVX Potato virus X
• the 5 ' end has an m7Gppp cap and the 3' end is polyadenylated.
• a full-length cDNA clone of PVX has been constructed and infectious RNA transcripts obtained (Hemenway et al, 1990).
• a DNA-launching plasmid is constructed based on PVX RNA containing PVX cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and having a polyadenylation site at its 3 ' end.
• a convenient restriction site may also be included at the 3 ' end.
• a foreign gene may be expressed from an additional subgenomic RNA.
• Transgenic plants are obtained having one or more trans-acting factors fused to the
• a DNA-launching plasmid is constructed having a DNA-dependent RNA polymerase promoter, polyadenylation site, and the PVX cDNA sequence in which the ORF2
• Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator.
• Such factors may include protein A or capsid protein precursor alpha, and preferably will also include a movement protein from a plant virus, such as 30K of TMV or 35K of RCNMV.
• Trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
• Transgenically expressed trans-acting factors preferably lack at least one cis-acting factor which is necessary for their replication, such as the 5 ' and/or 3 ' end.
• a DNA-launching plasmid is constructed based on TSWV RNA M in which the Gl and G2 coding sequences are replaced with at least one foreign gene or sequence.
• Such DNA- launching plasmid contains a DNA-dependent RNA polymerase promoter and TSWV RNA M cDNA fused to the self-cleaving ribozymes at the 5' and 3' ends.
• a DNA- launching plasmid is constructed based on TSWV RNA S in which the N coding region is replaced with a foreign gene or sequence.

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• 1999
  • 1999-05-21 CA CA002329509A patent/CA2329509C/en not_active Expired - Lifetime
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