WO1993024626A1 - Production d'animaux transgeniques avec des spermatozoïdes transformes par voie biolistique - Google Patents

Production d'animaux transgeniques avec des spermatozoïdes transformes par voie biolistique Download PDF

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Publication number
WO1993024626A1
WO1993024626A1 PCT/US1992/004426 US9204426W WO9324626A1 WO 1993024626 A1 WO1993024626 A1 WO 1993024626A1 US 9204426 W US9204426 W US 9204426W WO 9324626 A1 WO9324626 A1 WO 9324626A1
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WO
WIPO (PCT)
Prior art keywords
spermatozoa
dna
medium
carrier particle
gel
Prior art date
Application number
PCT/US1992/004426
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English (en)
Inventor
Oleg Larionov
Vasily N. Dobrovolsky
Original Assignee
Scientific Dimensions Usa, 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 Scientific Dimensions Usa, Inc. filed Critical Scientific Dimensions Usa, Inc.
Priority to AU21460/92A priority Critical patent/AU2146092A/en
Priority to PCT/US1992/004426 priority patent/WO1993024626A1/fr
Publication of WO1993024626A1 publication Critical patent/WO1993024626A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to methods of creating transgenic animals which contain cloned genes taken up into spermatozoa such that the spermatozoa function as vectors when fertilizing an egg of a sufficiently related species.
  • the spermatozoa take up the cloned DNA, for example, as a result of being bombarded by DNA-laden particles from a gene gun while fixed to an agarose gel.
  • the cloned sequence is transmittable to offspring.
  • sperm cells Only recently has cloned DNA been introduced directly into spermatozoa [Lavitrano et al., Cell 57. 717-723 (1989)]. Indeed, mature mouse sperm cells incubated in an isotonic buffer with cloned DNA will capture DNA molecules. Thus, sperm cells may be used as vectors for introducing foreign DNA into eggs at fertilization. Mouse eggs can be fertilized in vitro by sperm incubated in the cloned DNA solution then transferred, at the two-cell stage, to the oviducts of foster mothers.
  • mice Approximately 30% of 250 screened mice revealed clear sequence homology to the cloned DNA sequence.
  • the key to this method is the capture of foreign DNA by spermatozoa through exposure in an isotonic solution.
  • Foreign genes have also been introduced into spermatozoa using liposomes, [Hofer, Europ. Biotech. Newsletter 47, 4704 (1988)].
  • the spermatozoa loaded by the liposome method have been used to transform egg or somatic cells under intra- or extracorporal conditions to produce hepatitis surface antigen and a rat immunoglobulin. [German Pat. No. WO 87/05325].
  • Transgenic animals also have been produced by introducing foreign
  • Dupont's PDS-2000 utilizes microparticles (usually made of tungsten or gold) laden with the cloned DNA to pierce a cell membrane and introduce the DNA into the cell.
  • microparticles usually made of tungsten or gold
  • Dupont's PDS- 2000 has been used on muscle cells, human cultured cells, fibroblasts, Chinese hamster ovaries and lymphocytes to cause the up-take of cloned DNA
  • the gene gun requires its target cells to be exposed on a support surface to prevent particle breaking in the medium and for efficient DNA placement.
  • Dupont's PDS-2000 The initial conditions for using Dupont's PDS-2000 vary, but Dupont suggests the following conditions to optimize bombardment: cells should be maintained at 15 inches of Hg, for as brief a period as possible (1- 3 min.) optimizing vacuum pressure up to 27 inches of Hg in three such integrals. This increases the speed of the microcarriers. If cells become damaged or cell survival decreases, vacuum levels should be reduced. In addition, as the distance between the accelerator and the stopping plate increases, the velocity of the microcarrier decreases, and dispersion increases. Thus, different ranges of distance should be tried for comparison. The microcarrier must be placed optimally in the acceleration tube so the velocity is optimized. Microcarriers should be one-tenth the diameter of the cell.
  • M5 average is 0.35 ⁇ m
  • M10 average is 0.73 ⁇ m
  • tungsten and the one ⁇ m gold particles have all been used successfully on animal cells in tissue culture [DuPont BiolisticTM PDS-1000 Instruction Manual]. The successful bombardment of live sperm has not been yet reported.
  • Figure 1 is a schematic diagram of a recombinant vector used to transfer sperm in a preferred embodiment of the present invention.
  • Figure 2 summarizes the various general steps of certain preferred embodiments of the invention.
  • Figure 3 is a sketch of the vector construction used on the BLG gene for human gamma interferon expressing in mammary gland of transgenic animals.
  • the purpose of this invention is to create a universal, simple, efficient and inexpensive method for production of transgenic live stock transmitting foreign genes to their progeny. This method will obviate the difficulties inherent with recovering early embryos and treating donor and acceptor animals.
  • a method of producing a transgenic animal where DNA which expresses a desired trait is bound to a carrier particle, the carrier particle is inserted into spermatozoa, and the spermatozoa is used to fertilize an unfertilized egg from a species sufficiently related to the one from which said spermatozoa was collected.
  • a gene gun can be used to insert a carrier particle bound with DNA into spermatozoa. Any method of inserting the DNA-laden carrier particle can be used, although bombardment with a gene gun is preferred.
  • the carrier particles to which the cloned DNA is bound are tungsten or gold. Any other material may be used to bind the cloned DNA if it will sufficiently survive the chosen insertion method and properly present the
  • Fertilization of the unfertilized egg by the spermatozoa containing the inserted DNA can be by in vitro fertilization, by artificial insemination (for example, in the oviduct), or by any other means for uniting genetic material from gametes.
  • a method for reversibly fixing sperm so that they may be successfully bombarded by DNA-laden particles is also disclosed.
  • Sperm recovered from epididymis is dissolved at room temperature in a small volume of medium, then dropped onto the surface of slightly dried 1% agarose gel. After liquid evaporation, the spermatozoa become fixed on the gel. The bombardment process can occur for three to five minutes, after which a medium is added to the gel so the spermatozoa can regain motility.
  • This invention in certain preferred embodiments, consists of the following steps (see fig. 2): 1) fixing spermatozoa onto agarose gel, 2) bombarding fixed spermatozoa (head dimensions are approximately 2x5 ⁇ m) by DNA-coated particles using a gene gun (gold or tungsten particles with diameter of 0.2-0.5 ⁇ m); 3) harvesting bombarded spermatozoa and using centrifugation in a Percoll density gradient to isolate spermatozoa containing DNA-coated particles; and 4) using isolated spermatozoa for artificial insemination. After fertilizing an egg with spermatozoa carrying DNA-coated particles, a male pronucleus will form, followed by dissociation of the foreign DNA from the particle surface and integration of it into the genome. The particle will be torn away during embryo cleavage.
  • a vector was constructed by amplifying [using polymerase chain reaction (PCR)] of the 3'-end of the beta-lactoglobulin (BLG) gene comprising the last intron and poly(A) site and inserting of that fragment into a Pvu II site (fig. 1). More specifically, the 11.3kb DNA fragment comprising the 4.9kb transcription unit, the 4.8kb 5' flanking sequence and the 1.6kb 3' flanking sequence of the BLG gene was cloned (using standard methods of genetic engineering [Davis et al., Basic methods in Molecular Biology. Elsivier, N.Y. (1986)] into plasmid ⁇ BRUC318 (fig.
  • PCR polymerase chain reaction
  • a vector was created for human gamma-interferon (g-IFN) expression in the mammary gland.
  • g-IFN human gamma-interferon
  • PCR polymerase chain reaction
  • two synthetic oligonucleotides Seq. Id. No:3 and Seq. Id. No:4 were used as primers. They contained recognition sequences for restriction endonucleases SnaBI and Hind HI at their 5' ends and were complementary to flanks of this fragment.
  • PCR conditions were: lOOpmol of each primer, O.lng of target DNA (BLG gene), 67mM TrisHCl pH8.8, 16.6mM (NH 4 ) 2 S0 4 , 6.7mM MgCl 2 , lOmM DTT, 0.2mM of each dNTP, lOO ⁇ g/ml BSA, Taq I DNA polymerase 2un, H 2 0 to final volume of lOO ⁇ l. A total of 30 cycles were performed, each cycle being 30 sec. at 95 °C, 30 sec. at 65 °C, 2 min. at 72 °C.
  • DNA is bound to tungsten or gold particles using the following procedure: 1.25 mg of tungsten is heated in 95% EtOH at 65°C for four hours. 25 ⁇ g of DNA, 2.5M CaCl 2 and 0.1M spermidine are combined to a final volume of 575 ⁇ l (final concentration of 1.1M CaCl 2 and 8.7mM spermidine). The mixture is vortexed at 4°C for 10 minutes, then centrifuged at a low speed (500 rpm) for 5 minutes. Approximately 550 ⁇ l of supernatant is then removed and the remaining 25 ⁇ l is separated into l ⁇ l aliquots for loading onto a macrocarrier. It is better to use gold particles as tungsten may be toxic. DNA is bound to gold particles according to the method described by Du Pont. [Newsletter, A Publication of the Du Pont Company, 1, 1-4 (1990)].
  • spermatozoa are bombarded by accelerated DNA-coated microparticles from a gene gun, such as the PDS- 2000.
  • spermatozoa are reversibly fixated on a low melting point agarose gel.
  • sperm is collected from the epididymis, and dissolved at room temperature in a small volume (approx. 50-150 ⁇ l) of M2 medium [Quinn, et al., J. Reprod. Fertil 66, 161-168 (1982). The sperm solution is then dropped onto the surface of a slightly dried 1% agarose gel (Sea Plaque agarose, FMC, USA).
  • the spermatozoa are fixed on the gel. Three to five minutes later, during which time bombardment with foreign DNA can occur, M2 medium (50-250 ⁇ l) is added to the gel to reverse fixation. About 60-80% of fixed spermatozoa regain motility in the liquid phase.
  • Spermatozoa containing DNA-coated particles are separated from other spermatozoa by density gradient centrifugation.
  • the density gradient is formed in 10- 15ml centrifuge tubes during high-speed centrifugation (15 min. 29000g) of Duldecco's PBS containing 60-90% of Percoll. Bombarded spermatozoa washed over the surface of the gel are layered onto preformed density gradient tubes and after low-speed centrifugation (20 min. 400g) at room temperature, they are separated on fractions depending on their floating density.
  • Spermatozoa which contain particles are higher density so they concentrate closer to the bottom of the tube. The desired spermatozoa are then collected by puncturing of the tube bottom.
  • the DNA-laden spermatozoa are then used to artificially inseminate a female specimen or to fertilize eggs by other known means.
  • the preferred method of fertilization is to introduce DNA-laden spermatozoa directly into the oviduct of pseudopregnant females (females mated by sterile males) or transfer the selected spermatozoa (from density gradient tube) to the the oviduct or the uterus using fine glass pipet (outer diameter 100-120 ⁇ m)
  • This procedure is analogous to the procedure of embryo transfer into the uterus [Hogan et al., Manipulating the Mouse Embryo. (Cold Spring Harbor Laboratory, N.Y., 142-145 (1986)]).
  • the normal artificial insemination of a mouse female is difficult due to particularities of organization of mouse female reproductive organs. In vitro fertilization is also possible.
  • oligonucleotides e.g., Seq. Id. No:5 and Seq. Id. No:6 are used as primers for checking the integration of the g-IFN cDNA. They are complementary to g-IFN cDNA and form 252bp DNA fragments during PCR.
  • PCR conditions 20pmol of each primer, 0.1-0.5 ⁇ g of mouse genomic DNA 0.2mM of each primer, 50mM KC1, lOmM TrisHCl pH8.14, 1.5mM MgC- 2 , lOO ⁇ g/ml gelatin, Taq I DNA polymerase 1-1.5 units, and H 2 0 to final volume of 20 ⁇ l.
  • the mixture runs for 30 cycles, each being: 30sec 94°C, 30sec 55°C, 30sec 72°C. 10-15 ⁇ l aliquots of reaction mix is electrophoresed on 1.5% agarose gel in the presence of ethidium bromide at 100V for 30min and viewed under UV light.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

Procédé de production d'animaux transgéniques, consistant à lier à une particule de l'ADN qui exprime un caractère désiré, à insérer dans des spermatozoïdes la particule vecteur avec l'ADN lié, et à fertiliser un ÷uf suffisamment apparenté. L'invention décrit également un procédé de fixation réversible des spermatozoïdes sur un gel d'agarose légèrement séché, de manière à ce qu'ils puissent être bombardés par des particules chargées d'ADN au moyen d'un fusil genétique.
PCT/US1992/004426 1992-05-28 1992-05-28 Production d'animaux transgeniques avec des spermatozoïdes transformes par voie biolistique WO1993024626A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU21460/92A AU2146092A (en) 1992-05-28 1992-05-28 Transgenic animal production with biolistically transformed spermatozoa
PCT/US1992/004426 WO1993024626A1 (fr) 1992-05-28 1992-05-28 Production d'animaux transgeniques avec des spermatozoïdes transformes par voie biolistique

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1992/004426 WO1993024626A1 (fr) 1992-05-28 1992-05-28 Production d'animaux transgeniques avec des spermatozoïdes transformes par voie biolistique

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WO1993024626A1 true WO1993024626A1 (fr) 1993-12-09

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2282139A (en) * 1993-09-24 1995-03-29 Univ Reading Introducing DNA into the germ line of birds
WO1999049066A1 (fr) * 1998-03-23 1999-09-30 Ralf Schnabel Methode de transformation balistique de caenorhabditis elegans
US7053187B2 (en) 2000-03-28 2006-05-30 Gioagri Corporation Sperm-specific monoclonal antibody, mAbC
US7067308B1 (en) 2000-03-28 2006-06-27 Bioagri Corporation Vector for genetically modifying non-human animals
US9150881B2 (en) 2009-04-09 2015-10-06 Proteovec Holding, L.L.C. Production of proteins using transposon-based vectors
US9150880B2 (en) 2008-09-25 2015-10-06 Proteovec Holding, L.L.C. Vectors for production of antibodies
US9157097B2 (en) 2008-09-25 2015-10-13 Proteovec Holding, L.L.C. Vectors for production of growth hormone

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005325A1 (fr) * 1986-03-03 1987-09-11 Transgene Gmbh Procede pour transferer des substances organiques et/ou inorganiques a des cellules-oeufs et/ou a des cellules somatiques d'animaux, et compositions utilisees a cet effet
WO1990003439A1 (fr) * 1988-09-22 1990-04-05 Amgen Inc. Procede de transfert de genes dans les poulets et autres especes aviennes
WO1990008192A1 (fr) * 1989-01-10 1990-07-26 Consiglio Nazionale Delle Ricerche Technique d'introduction d'adn exogene dans des cellules animales somatiques et germinatives
WO1990008832A1 (fr) * 1989-01-27 1990-08-09 National Research Development Corporation Vecteur retroviral et son utilisation dans la production d'animaux transgeniques
WO1991000359A1 (fr) * 1989-06-26 1991-01-10 Agracetus, Inc. Transformation au moyen de particules de cellules somatiques animales
WO1991007487A1 (fr) * 1989-11-16 1991-05-30 Duke University Transformation de cellules tissulaires animales a l'aide de particules
EP0431839A1 (fr) * 1989-12-03 1991-06-12 Scopus-Genetics (Israel) Ltd. Solution tampon
WO1991018991A1 (fr) * 1990-05-29 1991-12-12 E.I. Du Pont De Nemours And Company Procede et appareil ameliores d'introduction de substances biologiques dans des cellules vivantes
WO1991019781A1 (fr) * 1990-06-21 1991-12-26 Agracetus, Inc. Appareil de transformation genetique

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1987005325A1 (fr) * 1986-03-03 1987-09-11 Transgene Gmbh Procede pour transferer des substances organiques et/ou inorganiques a des cellules-oeufs et/ou a des cellules somatiques d'animaux, et compositions utilisees a cet effet
WO1990003439A1 (fr) * 1988-09-22 1990-04-05 Amgen Inc. Procede de transfert de genes dans les poulets et autres especes aviennes
WO1990008192A1 (fr) * 1989-01-10 1990-07-26 Consiglio Nazionale Delle Ricerche Technique d'introduction d'adn exogene dans des cellules animales somatiques et germinatives
WO1990008832A1 (fr) * 1989-01-27 1990-08-09 National Research Development Corporation Vecteur retroviral et son utilisation dans la production d'animaux transgeniques
WO1991000359A1 (fr) * 1989-06-26 1991-01-10 Agracetus, Inc. Transformation au moyen de particules de cellules somatiques animales
WO1991007487A1 (fr) * 1989-11-16 1991-05-30 Duke University Transformation de cellules tissulaires animales a l'aide de particules
EP0431839A1 (fr) * 1989-12-03 1991-06-12 Scopus-Genetics (Israel) Ltd. Solution tampon
WO1991018991A1 (fr) * 1990-05-29 1991-12-12 E.I. Du Pont De Nemours And Company Procede et appareil ameliores d'introduction de substances biologiques dans des cellules vivantes
WO1991019781A1 (fr) * 1990-06-21 1991-12-26 Agracetus, Inc. Appareil de transformation genetique

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2282139A (en) * 1993-09-24 1995-03-29 Univ Reading Introducing DNA into the germ line of birds
WO1999049066A1 (fr) * 1998-03-23 1999-09-30 Ralf Schnabel Methode de transformation balistique de caenorhabditis elegans
US6433247B1 (en) 1998-03-23 2002-08-13 Devgen N.V. Ballistic transformation of C. elegans
US7053187B2 (en) 2000-03-28 2006-05-30 Gioagri Corporation Sperm-specific monoclonal antibody, mAbC
US7067308B1 (en) 2000-03-28 2006-06-27 Bioagri Corporation Vector for genetically modifying non-human animals
US8148143B2 (en) 2000-03-28 2012-04-03 Kwang-Hua Development And Investment Ltd. Method and composition for genetically modifying non-human cells and animals
US9150880B2 (en) 2008-09-25 2015-10-06 Proteovec Holding, L.L.C. Vectors for production of antibodies
US9157097B2 (en) 2008-09-25 2015-10-13 Proteovec Holding, L.L.C. Vectors for production of growth hormone
US9150881B2 (en) 2009-04-09 2015-10-06 Proteovec Holding, L.L.C. Production of proteins using transposon-based vectors

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