EP0644945A4 - Die vorbehandlung von mikroprojektilen von ihrer verwendung in einer partikelpistole. - Google Patents

Die vorbehandlung von mikroprojektilen von ihrer verwendung in einer partikelpistole.

Info

Publication number
EP0644945A4
EP0644945A4 EP93904738A EP93904738A EP0644945A4 EP 0644945 A4 EP0644945 A4 EP 0644945A4 EP 93904738 A EP93904738 A EP 93904738A EP 93904738 A EP93904738 A EP 93904738A EP 0644945 A4 EP0644945 A4 EP 0644945A4
Authority
EP
European Patent Office
Prior art keywords
beads
dna
tungsten
particles
nitric acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP93904738A
Other languages
English (en)
French (fr)
Other versions
EP0644945A1 (de
Inventor
Dwight Tomes
Margit Ross
Leigh Bangs
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pioneer Hi Bred International Inc
Original Assignee
Pioneer Hi Bred International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pioneer Hi Bred International Inc filed Critical Pioneer Hi Bred International Inc
Priority claimed from CA002132517A external-priority patent/CA2132517C/en
Publication of EP0644945A1 publication Critical patent/EP0644945A1/de
Publication of EP0644945A4 publication Critical patent/EP0644945A4/de
Ceased legal-status Critical Current

Links

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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection

Definitions

  • This invention relates to an improved process for transferring biological materials such as nucleic acid into the cytoplasm of living cells.
  • biological materials such as nucleic acid into the cytoplasm of living cells.
  • Such materials can include biological stains, proteins (antibodies or enzymes), and, most commonly, nucleic acids genetic material (either RNA or DNA).
  • Most of the techniques used are painstakingly slow and use methods which transport materials into, at most, only a few cells at a time.
  • An earlier invention of one of the joint inventors, Dwight Tomes, relates to an improved particle gun which uses a gun having a rifled barrel.
  • the disclosure of particle gun bombardment and the method of transport of biological materials such as DNA into living cells as described in Tomes, IMPROVED PARTICLE GUN, filed May 12, 1989, Serial No. 07/351,075, is incorporated herein by reference.
  • the effectiveness of particle transport is, of course, measured by the ability of living cells into which the transported particles have been inserted to pick up and express the biological material. This, of course, depends upon a wide variety of conditions. The less the expression, the less successful the transport. Correspondingly, the more successful the expression of the living cells, i.e. the extent that they pick up and express the transported biological material, the better the nucleic acid insertion experiments.
  • the biological material (DNA for example) is mixed with the carrier.
  • the carrier generally is comprised of a substantially inert metal in the form of small beads which function as microprojectiles.
  • the microprojectiles have a diameter within the range of about 1 micron to about 4 microns.
  • These beads can be made from tungsten, palladium, platinum or gold or an alloy thereof. Tungsten is preferred for reasons of economics. However, tungsten generally does not give as good or as successful transport and expression as does gold unless used with the parent pre-treatment process. Beads can generally range from about 1 micron to about 1.5 microns in diameter.
  • the beads are mixed with a small amount of biological material such as DNA or RNA. This is mixed with calcium chloride and a certain amount of polyamine is added.
  • tungsten particles at a concentration of 15-400mg in 2ml sterile water are placed in a sterile 10 ml centrifuge tube and agitated to suspend the beads.
  • the preferred amount of beads is 375mg/2ml.
  • Twenty-five ⁇ l of the suspended tungsten beads are placed in an Eppendorf tube and DNA is added at a concentration of 1 ⁇ g/ ⁇ l with the amount varying between l ⁇ l and 20 ⁇ l, the preferred amount being lO ⁇ l.
  • a calcium chloride solution of 25/ ⁇ l and having a concentration between 1.0-4.0M, preferably 2.5M is mixed with the DNA/bead mixture.
  • spermidine While addition of spermidine to the biological material/microprojectile combination has been previously employed, it has now been more broadly discovered that addition of a variety of polyammines to mixtures of tungsten beads and DNA or RNA in preparing microprojectiles significantly improve rates of transformation. While not intending to be limited by theory, it is believed that the polyamine improves delivery of biological material to the cells in this process by improving adherence of the materials to the microprojectile beads. Suitable polyammines have been found to include, for example, spermine, spermidine, caldine, thermine, and the like. The preferred polyamine, spermine, has been found to be superior to the previously disclosed spermidine additive.
  • a polyamine preferably spermine
  • a concentration between 0.05M and 0.5M, preferably at 0.1M is added followed by finger vortexing.
  • This mixture is allowed to stand for 10 minutes prior to centrifugation for one to two minutes at 9,000 rpm. Centrifugation may be used but is not required.
  • the microprojectile mixture forms a pellet at the bottom of the Eppendorf tube. Before use, supernatant is withdrawn from the tube to provide a final volume of 30 ⁇ l.
  • the DNA/bead mixture is sonicated briefly to suspend the microprojectiles prior to use.
  • the suspended microprojectiles carrying the biological material are transferred to the forward end of the macroprojectile by micropipette in aliquots of l-5 ⁇ l, with 1.5 ⁇ l preferred.
  • the invention relates to an improvement in the process of successfully transporting biological material into living cells by bombarding the cells with biological material coated particles.
  • the preferred particles are tungsten and the method of the present invention allows use of the more economical tungsten beads rather than gold.
  • the achievement of the objectives of the present invention is accomplished by pretreating the preferred tungsten beads with a strong inorganic acid, preferably nitric acid.
  • a strong inorganic acid is selected from the group consisting of nitric acid, sulfric acid, hydrochloric acid and phosphoric acid or mixtures thereof.
  • the preferred acid is nitric acid.
  • the concentration of the strong inorganic acid is not critical. At the lower end, a concentration of as low as .01 molar has been used successfully and at the upper end there appears to be only a process economic consideration. Namely excessive amounts are not harmful to the metallurgy or surface of the beads, but there is not reason to use concentrations beyond an amount sufficiently concentrated to effectively clean and pretreat the beads.
  • the upper limit would be a practical limit of about 1.0 molar, preferably the concentration of the acid would be within the range of 0.1 molar to about 0.5 molar.
  • the time of treatment is not critical but generally can range from about 5 minutes to about 60 minutes, preferably from about 10 minutes to about 20 minutes.
  • the importance of time is sufficient time for the beads, to successfully contact the nitric acid.
  • successful contact is best achieved when beads are agitated during their pre- treatment.
  • the agitation is continuous and most preferably the agitation is by sonication.
  • the particles, if treated with nitric acid should not settle out of the liquid suspension if they are clean.
  • the washed particles stayed in suspension, thus it was determined that the 20 minute wash was sufficient for successfully cleaning the tungsten particles. Prior to the nitric acid wash, tungsten immediately fell out of suspension.
  • the precise beads employed are not critical and the process shows distinct advantages when the beads are any beads selected from the group consisting of tungsten, palladium, platinum and gold or an alloy thereof.
  • the beads Preferably have a diameter of from about 1 micron to about 1.5 micron.
  • the most preferred beads are tungsten since by using the pre- treatment of the present invention tungsten can be used as effectively as more expensive metals such as platinum, palladium and gold.
  • the acid is rinsed from the beads, and the rinse may be a water rinse, for example followed by an alcoholic rinse, for example, followed by ethanol.
  • the beads are then conventionally air dried, perhaps by vacuum speed drying.
  • the biological material such as DNA is added to the microprojectiles and proper mixing is done.
  • Conventional additives is know to be mixed with a biological material such as calcium chloride and a certain amount of amines such as a polyamine.
  • the plant tissue is bombarded to achieve the transport of biological material into living cells or simulation therein.
  • the following examples are offered to illustrate but not necessarily limit the present invention. They demonstrate that with the pretreatment of the present invention tungsten is more effective than gold beads.
  • the purpose was to evaluate the effect of a nitric acid cleaning on tungsten and gold particles prior to drying them in a speed vacuum. They were compared to a standard control.
  • the genotype material used was an embryonic maize suspension of DNA.
  • the maize suspension medium contained Murashige and Skoog salts: Reference: Murashige, T. , Skoog, F. : A Revised Method for Rapid Growth and Bioassays with Tobacco Tissue Cultures, Physiol. Plant. 15:473-497 (1962) with 2.0mg/L of 2,4-D, which is 2,4-Dichorophenoxy-acetic Acid and 3% sucrose.
  • the media used involved maize callus medium with the following description: AT salts with 700mg/L proline, 0.75mg/L 2,4-D, 3% sucrose.
  • the particle gun treatment involved use of the particle gun as described previously in the incorporated by referenced Tomes application.
  • the beads were tungsten 1.8um from GTE, tungsten 1.2um from GE, gold ( flakeless) from Engelhard Industries, 25mg tissue per plate being used. There was a 0.25M mannitol pretreatment, overnight, and there was one bombardment per sample using pDP460 DNA which contains the GUS gene. pDP460 is described in Example 2 below.
  • the bombardment occurred in the following way. Suspension cells were used one day after subculture and sieved through a 710um screen and resuspended in maize suspension medium + 0.25M mannitol at 50mg/ml (lg tissue in 20ml medium). They were agitated on the shaker overnight. To ready for particle gun treatment, 0.5ml aliquots were pipetted onto the center of double layers of 617 Whatman grade filter paper to which 1ml maize suspension medium + 0.25M mannitol has been added. The cells were concentrated in the center of each plate to maximize exposure to bombardment. Six repetitions were used for all particle treatments including six repetitions for the standard positive control. Twenty-six samples in total were used. Two independent repetitions of the experiment were completed.
  • Beta-glucuronidase cytochemical analysis for transient gene expression completed 36 hours post treatment.
  • Genotype 54-68-5, maize e bryogenic suspension
  • EXAMPLE 2 In this example, the process of the present invention, using tungsten pretreated with nitric acid was compared with tungsten beads not pretreated with nitric acid. It also compared with bombardment of cells using non-nitric acid treated tungsten microprojectiles in a particle gun, using the stan- dard protocol recommended and publicly published by DuPont.
  • the DuPont protocol, as published, is as follows:
  • the Pioneer tungsten/DNA precipitation method is as follows: add lO ⁇ l (at l ⁇ g/ ⁇ l) DNA to speed vacuum dried tungsten, mix with pipettor. Add 25 ⁇ l of 2.5M CaClnose to tungsten/DNA suspension, mix with pipettor. Add lO ⁇ l to 0.1M spermine to tungsten/DNA/CaCl suspension, finger vortex. Allow suspension to stand at room temperature for 15 minutes, then withdraw 15 ⁇ l of supernatent. Sonicate suspension prior to aliquoting 1.5 ⁇ l aliquots onto macroprojectiles. The embroyogenic maize suspension 2-122-4 (W23 x B73 ) was used. The culture media and particle bombardment procedures were as outlined in example 1 (see page 9) .
  • the particle preparations prepared on day 1 by the respective methods were sequentially sampled for experiments on days 1, 2, 3, 4, and 5 using particles on day 1. DNA/particle mixing using the respective procedures was done on each day. Sixteen samples for each set of this example were completed. Fourteen were treated with pDP460 DNA.
  • the plasmid pDP460 contains an enhanced promoter spanning nucleotides - 421 to +2 of CaMV 35S [R.C. Gardner et al. Nucleic Acids Res. 9, 2871 (1981)], a 79-bp Hind Ill-Sal I fragment from pJIHOl spanning the 5' leader sequence of tobacco mosaic virus [D.R. Gallie, D.E. Sleat, J.W. Watts, P.C.
  • the data of significance in Table 2 is the data under ratio of "INV” to DuPont. There, it is shown that the GUS gene (pDP460) is successfully delivered and expressed in the cells, as measured by the cells turning blue in a much higher ratio, with the process of the present invention than with the standard DuPont process which utilizes similar tungsten particles, but does not pretreat with nitric acid.
  • Example 2 a comparison of the process of the present invention using nitric acid pretreated beads in the manner described in Example 2 and using tungsten beads which were not pretreated according to the standard DuPont protocol, were prepared precisely as above described.
  • the example was run to show not only successful transport into the cells, but whether or not the DNA after transport is integrated into plant DNA such that cell division carrying the foreign DNA occurs. In other words, can you not only transfer the DNA into the cells, but having done that, can you successfully grow up a plant, which has the foreign DNA integrated into the plant DNA.
  • standard procedure prenitric acid treated tungsten beads were used with the plant material being Xanthi tobacco cotyledons. The DNA expressed was pDP456 [NPTII+GUS] .
  • the plasmid pDP456 contains two different plant transcription units (PTU's).
  • the first PTU includes an enhanced promoter spanning nucleotides -421 to +2 of CaMV 35S with the region from -421 to -90 duplicated in tandem [R.C. Gardner et al., Nucleic Acids Res. 9, 2871 (1981)], a 79-bp Hind Ill-Sal I fragment from pJIIlOl spanning the 5' leader sequence of tobacco mosaic virus [D.R. Gallie, D.E. Sleat, J.W. Watts, P.C. Turner, T.M.A. Wilson Nucleic Acids Res.
  • the second PTU is identical to the first, except that it includes the NPTII coding sequence [R.T. Frayley et al. , Proc. Natl. Acad. Sci. U.S.A. 80, 4803 (1983)] in place of GUS.
  • NPTII coding sequence R.T. Frayley et al. , Proc. Natl. Acad. Sci. U.S.A. 80, 4803 (1983)] in place of GUS.
  • four in vitro grown Xanthi cotyledons were prepared and bombarded as described in Tomes et al. Plant Mol. Biol. 14: 261- 268 (1990).
  • microprojectile/DNA DuPont comparison treatments were made using the earlier described standard DuPont protocol.
  • the treatments of the present invention were as described in the previous example. All DNA samples received one bombardment by the particle gun. After particle gun treatment, all DNA treated samples were transferred to a selection medium as described in the Tomes, et al. publication.
  • Table 3 indicates NPTII enzyme assays confirmed stable integration of pH1456 in each sample noted as NPTII+. Table 3 shows the assay results for independent trans-genes from the process protocol or the DuPont protocol.
  • Example 4 was run to confirm if nitric acid treatment resulted in an increase in transformation with the DuPont protocol compared with the protocol of the present invention.
  • the material used was genotype 2-122-4 (W23 x 3) embryogenic maize suspension.
  • the DNA was pDP460.
  • the process of the present invention was employed using a nitric acid treatment and compared with the DuPont DNA/particle mixing protocol, and the DuPont protocol using nitric acid as the only variance.
  • the methodology was as Example 1.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
EP93904738A 1993-01-29 1993-01-29 Die vorbehandlung von mikroprojektilen von ihrer verwendung in einer partikelpistole. Ceased EP0644945A4 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CA002132517A CA2132517C (en) 1993-01-29 1993-01-29 Pretreatment of microprojectiles prior to using in a particle gun
CZ942328A CZ232894A3 (en) 1993-01-29 1993-01-29 Pretreatment of micro-projectiles prior their use in a particle gun
PCT/US1993/000817 WO1994017195A1 (en) 1993-01-29 1993-01-29 Pretreatment of microprojectiles prior to using in a particle gun

Publications (2)

Publication Number Publication Date
EP0644945A1 EP0644945A1 (de) 1995-03-29
EP0644945A4 true EP0644945A4 (de) 1997-01-15

Family

ID=27169854

Family Applications (1)

Application Number Title Priority Date Filing Date
EP93904738A Ceased EP0644945A4 (de) 1993-01-29 1993-01-29 Die vorbehandlung von mikroprojektilen von ihrer verwendung in einer partikelpistole.

Country Status (4)

Country Link
EP (1) EP0644945A4 (de)
JP (1) JPH07504575A (de)
SK (1) SK115094A3 (de)
WO (1) WO1994017195A1 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6406852B1 (en) * 2000-06-22 2002-06-18 Council Of Scientific And Industrial Research Method for preparation of microprojectiles for efficient delivery of biologicals using a particle gun

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4101309A (en) * 1976-10-13 1978-07-18 Tokyo Shibaura Electric Co., Ltd. Method for preparing tungsten materials
EP0397413A1 (de) * 1989-05-12 1990-11-14 Pioneer Hi-Bred International, Inc. Partikel-Gewehr
EP0486233A2 (de) * 1990-11-14 1992-05-20 Pioneer Hi-Bred International, Inc. Pflanzentransformationverfahren durch Verwendung von Agrobakterium-Arten

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3236634A (en) * 1962-02-12 1966-02-22 Jr Foraker Lambdin Process for production of high surface area tungsten and tungsten trioxide powders
GB1461176A (en) * 1974-04-11 1977-01-13 Plessey Inc Method of producing powdered materials
US4945050A (en) * 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
EP0197271B1 (de) * 1985-03-04 1989-04-19 Kabushiki Kaisha Toshiba Verfahren zur Herstellung hochreiner Molybdän- oder Wolframpulver in Form ihrer Metalle oder Oxide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4101309A (en) * 1976-10-13 1978-07-18 Tokyo Shibaura Electric Co., Ltd. Method for preparing tungsten materials
EP0397413A1 (de) * 1989-05-12 1990-11-14 Pioneer Hi-Bred International, Inc. Partikel-Gewehr
EP0486233A2 (de) * 1990-11-14 1992-05-20 Pioneer Hi-Bred International, Inc. Pflanzentransformationverfahren durch Verwendung von Agrobakterium-Arten

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SANFORD: "The biolistic process", TRENDS IN BIOTECHNOLOGY, vol. 6, December 1988 (1988-12-01), pages 299 - 302, XP002017086 *
See also references of WO9417195A1 *

Also Published As

Publication number Publication date
WO1994017195A1 (en) 1994-08-04
EP0644945A1 (de) 1995-03-29
SK115094A3 (en) 1995-07-11
JPH07504575A (ja) 1995-05-25

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