EP1111991A1 - Procede de mise en oeuvre de transgenese - Google Patents

Procede de mise en oeuvre de transgenese

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Publication number
EP1111991A1
EP1111991A1 EP99942164A EP99942164A EP1111991A1 EP 1111991 A1 EP1111991 A1 EP 1111991A1 EP 99942164 A EP99942164 A EP 99942164A EP 99942164 A EP99942164 A EP 99942164A EP 1111991 A1 EP1111991 A1 EP 1111991A1
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EP
European Patent Office
Prior art keywords
oocyte
nucleus
dna
spermatozoon
microinjection
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EP99942164A
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German (de)
English (en)
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EP1111991A4 (fr
Inventor
Anthony C.F. Perry
Teruhiko Wakayama
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Individual
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Individual
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Publication of EP1111991A1 publication Critical patent/EP1111991A1/fr
Publication of EP1111991A4 publication Critical patent/EP1111991A4/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
    • 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
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0335—Genetically modified worms
    • 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
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0338—Genetically modified Crustaceans
    • 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
    • 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
    • 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

  • Transgenesis is a process that results in the modification of genomes to carry newly-introduced DNA sequences.
  • the process commonly entails the genomic integration of foreign, or transgene (tg), DNA sequences.
  • the DNA sequences may encode desired characteristics, so that any transgenic animal (or plant) now carrying the genomic modification may possess one or more new characteristics endowed by it.
  • genomic modifications are transmissible through the germline, such that they may be transmitted to offspring, and thereafter vertically along the lineage of the transgenic organism.
  • transgenic animals and plants have great potential utility in agriculture, medicine and the production of bioactive compounds (neutraceuticals and pharmaceuticals). For example, the production of transgenic pigs expressing human major histocompatibility proteins on the surfaces of their cells would contribute to the usefulness of these animals in xenotransplantation.
  • Transgenesis by pronuclear microinjection does not as yet permit the outcome of tg insertion to be controlled or predicted due to the quasi-random nature of integration site and number of copies integrated into the host genome.
  • Greater control over the outcome of integration can be achieved by using (mouse) embryonic stem (ES) cell lines transfected with constructs capable of genome-targeted, homologous recombination.
  • Transfected ES cell lines can be selected and characterized in vitro to confirm the construct integration site. Reconstitution of embryos with such gene-targeted ES cells may then be used to produce chimaeric offspring. This method of genome modification is currently restricted to the one species for which established, germline-contributing ES cells exist - the mouse - with no demonstrated application to other species.
  • sperm heads are able to support full
  • membrane-disrupted spermatozoa are also able to support full development (Perry, A.C.F., Wakayama, T. & Yanagimachi, R. Biology of Reproduction 60, 747 [1999]). Structures underlying the membranes of spermatozoa
  • transgenic animals from a variety of different species; its flexibility permits the introduction of a broader spectrum of different types of tg and other molecules; it
  • the invention provides methods for introducing transgene (tg) nucleic acid (NA) into the cells of an organism, such as a plant or animal, or in vitro cultured cells, by injecting tg NA, or co-injecting tg NA that had previously been mixed with a nucleus, into an immature oocyte, an unfertilized oocyte or enucleated oocyte (known hereafter as "oocyte").
  • tg transgene
  • NA nucleic acid
  • unfertilized oocytes are those arrested at the metaphase ⁇ (met LI) of meiosis.
  • Met II oocytes are at the stage that normally participate in fertilization in mammals.
  • the invention further provides for the introduction of tg NA into a cell containing resident nuclear components, followed by activation of development.
  • transgene NA is intended to encompass any nucleic acid or its derivative that may be introduced into an oocyte to induce a change in the genomic sequence that was hitherto native to the host genome, thereby altering that genome.
  • the tg NA is deoxyribosenucleic acid, DNA.
  • nucleus used herein refers to the entire nucleus or that portion of the nucleus necessary for full embryonic development to term and beyond. In one preferred embodiment, the nucleus is the nucleus of a sperm cell.
  • the invention further provides methods for producing a transgenic animal by co-inserting tg NA with a nucleus.
  • the nucleus is exposed to the tg NA by, for example, mixing them together.
  • the nucleus is the nucleus of a spermatozoon whose membranes have been removed or disrupted. The invention allows for a variety of procedures of membrane disruption.
  • the nucleus is inserted into the oocyte by microinjection, and more preferably by piezo-electrically actuated microinjection.
  • a piezo-electrically actuated facilitates the microinjection process, rendering it swifter. This reduces cellular trauma, enhancing embryonic survival rate.
  • the cell reconstituted in this way may be permitted to develop. In one embodiment, development produces a relatively homogeneous populations of cells (for example stem cells). In a further embodiment, the reconstituted cell is allowed to develop into a blastocyst following culture in vitro, and the resulting embryo may be transferred to a suitable surrogate mother at this or a previous stage in embryonic development, to permit full development.
  • damage to membranes allows tg DNA to gain access to sub-nuclear elements, including but not limited to the perinuclear matrix (in the case of spermatozoa), the nuclear matrix, chromatin and genomic DNA.
  • the tg NA is a linear DNA fragment that encodes a readily detectable phenotypic marker.
  • Transgenic embryos and offspring resulting from co-insertion of a nucleus with such DNA possess a genomic alteration that may alter their characteristics (phenotype) in a manner that is readily detectable.
  • readily detectable markers that are suitable include firefly luciferase (Luc), Escherichia coli ⁇ -galactosidase (LacZ) and the Aequo ⁇ a victoria green fluorescent protein (GFP).
  • tg NA is mixed with the nucleus prior to co-injection using a micropipette.
  • co-injection is into an oocyte arrested at metaphase II (met LI) of meiosis.
  • metal LI metaphase II
  • GFP Aequoria victoria green fluorescent protein
  • LacZ Escherichia coli ⁇ -galactosidase
  • tg NA corresponds to an artificial chromosome such as a mammalian, yeast or bacterial artificial chromosome (MAC, YAC and BAC respectively: Schindelhauer, D. Bioessays 21, 76 [1999]; Peterson, K.R. Methods in Enzymology 306, 186 [1999]; Kim, U.J., Birren, B.W., Slepak, T., Mancino, V., Boysen, C, Kang, H.L., Simon, M.I. & Shizuya, H. Genomics 34, 213 [1996]).
  • an artificial chromosome such as a mammalian, yeast or bacterial artificial chromosome (MAC, YAC and BAC respectively: Schindelhauer, D. Bioessays 21, 76 [1999]; Peterson, K.R. Methods in Enzymology 306, 186 [1999]; Kim, U.J., Birren, B.W., Slepak, T., Mancino, V
  • tg NA corresponds to ribosenucleic acid (RNA) such as messenger RNA (mRNA), or to RNA-DNA heteroduplexes (chimasras that possess at least one mismatch) or to peptide nucleic acids.
  • RNA ribosenucleic acid
  • mRNA messenger RNA
  • RNA-DNA heteroduplexes chimasras that possess at least one mismatch
  • peptide nucleic acids peptide nucleic acids
  • the invention provides an efficient method for producing transgenic offspring by the co-injection of a nucleus with tg DNA into an unfertilized oocyte.
  • the invention is applicable to all organisms and collections of differentiated cells and stem cells, which can, or might be, generated following the insertion of a nucleus into an unfertilized oocyte.
  • This includes the nuclei of cells derived from, without limitation, amphibians, fish, birds (eg domestic chickens, turkeys, geese and the like) and mammals, such as primates, ovines, bovines, porcines, ursines, caprines, felines, canines, equines, murines and the like.
  • the nuclei are from spermatozoa.
  • the germane properties of sperm nuclei are conserved (see Kimura, Y., Yanagimachi, R., Kuretake, S., Bortkiewicz, H., Perry, A.C.F. & Yanagimachi, H. Biology of Reproduction 58, 1407 [1998]).
  • the co-introduction of tg DNA/nuclear material by microinjection is spatiotemporally distinct from methods that require cell fusion promoted either artificially or via in vitro (Lavitrano, M., Camaioni, A., Fazio, V.M., Dolci, S., Farace, M.G. & Spadafora, C. Cell 57, 717 [1989]).
  • the microinjection method entails first the selection of a nucleus (and NA) and subsequently its deposition into an oocyte by puncturing the plasma membrane of the oocyte.
  • Co-injection of tg DNA and nucleus by the method of the invention does not require that the nucleus be obtained from a living cell. This further distinguishes the method of the invention from claims exemplified by those wherein live sperm were mixed with tg DNA (in vivo or in vitro) and used to introduce the DNA via fertilization. Furthermore, co-injection of tg DNA and a nucleus from a membrane-disrupted cell according to the method of the invention, allows for the precisely controlled co-introduction of reagents that might be efficacious to the outcome of the procedure. Such a reagent may include an enzyme, antibody, or pharmacological signal transduction inhibitor that modulates recombination and/or embryo development to promote transgenesis. The introduction of the reagent into the oocyte may take place prior to, during, or after the co-introduction of tg NA and nucleus. BRIEF DESCRIPTION OF DRAWINGS
  • Figure 1 shows representative sagittal sections through the heads of mouse spermatozoa that were either intact ('fresh')(A), or whose membranes had been disrupted by triton X-100 (B), freeze-thawing (C) or freeze-drying (D) as per Example 1.
  • Abbreviations are: ac, acrosomal cap; eq, equatorial segment; pa, postacrosomal region. Plasma and acrosomal membranes (except for those in the equatorial region) are absent or disrupted. Disruption is clearest in the membranes of the acrosomal cap.
  • FIG. 2 shows transgenic embryos produced by single-shot double transgenesis.
  • Oocytes were microinjected with spermatozoa that had been preincubated with a mixture of pCX- LacZ and pCX-EGFP tg DNAs as per Example 3.
  • Panels show the same embryos (x400) after 3.5 days viewed by Hoffman modulation contrast microscopy (A) unstained, (B) for GFP expression under long-wavelength (480 nm) UV light, and (C) stained with X-Gal for ⁇ - galactosidase expression.
  • A unstained
  • B for GFP expression under long-wavelength (480 nm) UV light
  • C stained with X-Gal for ⁇ - galactosidase expression.
  • Figure 3 shows an analysis of tail-tip biopsies from transgenic founders and non-
  • transgenic controls Fluorescent stereomicroscopy (x40) of tail tips from non- transgenic (a; mouse #16) and transgenic, green-fluorescent (b; mouse #3) lines.
  • the present invention describes a distinctive set of methods for generating cells harboring an integrated transgene.
  • the method of the invention comprises the steps of: (I) Exposure of exogenous, tg NA to nuclear components (a nucleus, or portion thereof, including the chromosomes); (II) Microinjecting the tg NA-nucleus mixture or tg NA into an unfertilized egg; (III) Allowing the resulting cell to develop. Development may be to produce differentiated cells or stem cells to an embryo, or to an individual following embryo transfer into a surrogate mother. We now present the individual steps in greater detail and show how they are distinctively arranged in respect of one to the other in the present invention.
  • the method allows for exposure of exogenous, tg NA to nuclear components, prior to microinjection.
  • Nuclei may be from somatic or other cells.
  • Exogenous, tg NA may be introduced into cells by methods exemplified by, but not limited to, electroporation, lipofection, transfection, mixing or microinjection prior to the time of nucleus insertion. These methods are well known to those skilled in the art.
  • a fragment of pCX-EGFP DNA was mixed for 30-180 seconds on ice or 25°C by triturating with non-transgenic sperm whose membranes had been disrupted by treatment with a detergent, such as, but not limited to triton X-100 (3-[3-cholamidopropyl)dimethylammonio] 1-propanesulfonate) (CHAPS), sodium dodecylsulfate (SDS), sodium lauryl sulfate (SLS), mixed alkyltrimethylammonium bromide (ATAB), and the like.
  • sperm membrane disruption was by freezing/thawing or by freeze-drying. These methods cause sufficient damage to sperm to decapitate a proportion of the population. The extent of membrane damage increases in the order: freshly isolated sperm ⁇ triton X-100 ⁇ freeze/thaw ⁇ freeze-dry.
  • Tg NA may include single-stranded or double-stranded RNA or DNA, chimaeric heteroduplexes (such as RNA-DNA hybrids; see below) and relatively large molecular species such as chromosomes.
  • the NA may correspond to DNA molecules which are large (for example 50 kilobase pairs [kb] to >1 megabase pairs). Examples of large DNA molecules include, but are not limited to, artificial chromosomes such as mammalian, yeast and bacterial artificial chromosomes (MACs, YACs and BACs respectively).
  • the step of gentle mixing with a membrane-challenged sperm head likely allows stabilization of large DNA molecules by (i) allowing them to associate with the relatively massive protective structure of the sperm head, and (ii) not subjecting them to chemical or physical forces (such as shear forces) during the injection procedure, thereby increasing the success rate.
  • a further manner in which the method of the invention permits significant advantages over previous methods is that it provides means by which novel properties of the cytoplasm of the unfertilized (met II) oocyte may be harnessed.
  • nuclear decondensation following microinjection into met II oocytes provides a situation in which genomic DNA is relatively exposed and therefore more reactive (described in Perry, A.C.F., Wakayama, T. & Yanagimachi, R. Biology of Reproduction 60, 747 (1999).
  • the met II oocyte contains recombinogenic factors, since the injection of mouse met II oocytes with damaged and spermatozoa heated to 48*C results in recombination to produce bicentric (ie translocated), acentric, and ring chromosomes, and chromosome fragments (Ward, W.S., Perry, A.C.F., & Balhorn, R. Biology of Reproduction, accepted for publication).
  • the method of the invention thus provides for gene targeting via homologous recombination and/or by recruiting factors responsible for DNA repair, as, for example in chimaeroplasty (see below).
  • the invention allows for such recombination to be enhanced by the inclusion of site-specific and non-site-specific recombination-promoting agents in the nucleus-NA mixture.
  • agents include, without limitation, Escherichia coli RecA protein, the human RecA counterpart, HsDmc-1, single-stranded DNA binding proteins such as bacteriophage T4 gene 32 product, site specific recombinase(s) (such as Cre and Flp recombinases) and the like.
  • the NA used is a gene targeting DNA construct containing extensive sequences that match those of a small number (usually 1) of genomic loci native to the genome.
  • the design criteria for gene targeting vectors are well established and known by those skilled in the art.
  • these molecules are short ( ⁇ 100 nucleotides) RNA-DNA heteroduplexes (for example, Yoon, K., Cole-Strauss, A. & Kmiec, E.B. Proceedings of the National Academy of Sciences USA 93, 2071 [1996]) which contain the near-perfect compliment of a genomic sequence with the exception of a 1-3 base mismatch near the center.
  • RNA-DNA heteroduplexes for example, Yoon, K., Cole-Strauss, A. & Kmiec, E.B. Proceedings of the National Academy of Sciences USA 93, 2071 [1996]
  • Such molecules can direct cellular DNA repair machinery to introduce the mismatch into the genomic sequence.
  • the DNA repair machinery is within the oocyte, such that site-specific mutations may be introduced during, or following, nucleus-chimaeraplast co-injection into the met II oocyte, or injection of a chima ⁇ raplast in the absence of a nucleus.
  • the invention allows for the inclusion of additional substances during mixing. These may include, but are not limited to, modulators of nuclease activity (eg ethylenediamine tetraacetic acid [EDTA], aurintricarboxylic acid, restriction endonucleases and the like), apoptosis (eg aurintricarboxylic acid, and the like), proteolysis (eg leupeptin, E.64 and the like), and DNA binding proteins such as protamines and topoisomerases.
  • modulators of nuclease activity eg ethylenediamine tetraacetic acid [EDTA], aurintricarboxylic acid, restriction endonucleases and the like
  • apoptosis eg aurintricarboxylic acid, and the like
  • proteolysis eg leupeptin, E.64 and the like
  • DNA binding proteins such as protamines and topoisomerases.
  • supplementary agents are mixed with
  • the genome is provided by a nucleus already resident within the oocyte microinjected with the tg NA, as for example in the case of an enucleated met II oocyte that had (subsequent to enucleation) received the nucleus of a somatic cell via nuclear transfer, or a non-enucleated met LI oocyte.
  • These cells are activated to undergo development artificially using parthenogenetic agents (in the presence of cytokinesis blocking agents such as cytochalasin B, cytochalasin D and the like) by methods known to those skilled in the art.
  • Nucleus-NA mixtures are held for a period of time to allow nucleus-NA association. In one embodiment, the association is given at least 30 sec to occur. The mixture is then transferred to a microscope stage for microinjection. In a further embodiment, microinjection is completed within 1 h of nucleus exposure to NA.
  • Nuclei which have been exposed to tg NA are inserted into an unfertilized oocyte by microinjection.
  • Nucleus-NA mixtures are transferred to a droplet on the microscope stage of the microinjection unit such that they may be gathered into the microinjection needle for injection.
  • nucleus-NA mixtures are supplemented with a solution of polyvinylpyrrolidone to aid manipulation. Collection of the tg NA-nucleus sample to be injected is by aspiration into the injection pipette.
  • the microinjection needle is piezo-actuated.
  • a suitable piezo electric driving unit is sold by Prime Tech Ltd.
  • the unit is capable of transmitting a piezo-electric pulse to advance the microinjection pipette tip to which it is attached a very short distance (of the order of 0.5 ⁇ m) in a highly controlled and rapid manner.
  • the intensity and interval between each pulse (which may be varied on the control unit, with typical values of 1-5 for intensity and 1-16 for speed) are applied to advance the tip through the zona pellucida of the oocyte (whilst fixing the oocyte in place using light suction from a holding pipette).
  • the pipette tip is the apposed to the oocyte plasma membrane and advanced (toward the opposite face of the oocyte) until the oocyte plasma membrane is deeply invaginated.
  • a small number typically one
  • piezo pulses typically, intensity 1-4, speed, 1
  • the plasma membrane is punctured, allowing expulsion of tg NA-nucleus or tg NA into the cytoplasm of the oocyte.
  • injection is through a flush-ended borosilicate glass needle (of typical internal diameter, 4.5 - 10 ⁇ m) that contains mercury near its end; the mercury increases the momentum and control of the piezo- actuated needle tip.
  • Alternative microinjection variants may be used to insert the tg NA and/or nucleus, including conventional pipettes as exemplified in the description of Yanagida, K., Yanagimachi, R., Perreault, S.D. and Kleinfeld, R.G. Biology of Reproduction 44, 44 (1991).
  • Injection of nuclei may be into an oocyte from the same species, or from an oocyte taken from a different species.
  • the method of the invention allows for NA injection, or NA-nucleus coinjection, into oocytes, enucleated oocytes, or immature (eg germinal vesicle stage) oocytes, including pre-ovulatory oocytes that have been matured in vitro.
  • IMM In vitro maturation of oocytes (IVM) is desirable where sources of mature oocytes are limited or non-existent and may be in the presence of agents which render them more suitable for microinjection.
  • Bovine oocyte IVM has been described in WO 98/07841 and for mouse oocytes in Eppig & Telfer (Methods in Enzymology 225, 77, Academic Press [1993]). Mature oocytes may be obtained by inducing super-ovulation following the sequential administration of gonadotrophic or other hormones (for example, the sequential administration of human chorionic gonadotrophin and pregnant mare serum gonadotrophin) and the subsequent surgical harvesting of ova (eg 80-84 hours after the onset of estrous in the cat, 72-96 hours in the cow and 13-15 hours in the mouse).
  • gonadotrophic or other hormones for example, the sequential administration of human chorionic gonadotrophin and pregnant mare serum gonadotrophin
  • the subsequent surgical harvesting of ova eg 80-84 hours after the onset of estrous in the cat, 72-96 hours in the cow and 13-15 hours in the mouse.
  • the NA-exposed nucleus is from a diploid somatic cell, and is inserted into the cytoplasm of an oocyte whose chromosomes had been removed (an enucleated oocyte); the method of oocyte enucleation is well known to those skilled in the art and is utilized, for example, in Wakayama, T., Perry, A.C.F., Johnson, K., Zuccotti, M. & Yanagimachi, R. Nature 394, 369 (1998). Collection of nuclei is from cells that had been dispersed, for example by treatment with with a mixture of trypsin (0.025%) and ethylenediamine tetraacetic acid (EDTA; 0.75mM).
  • EDTA ethylenediamine tetraacetic acid
  • Cells are artificially stimulated to initiate development 0-6 h after reconstitution, using an stimulus such as, but not limited to, Sr 2+ , ethanol or an electric pulse, according to known methods.
  • the method of the invention applies to nuclei taken from the cells of amphibians, fish, birds (eg domestic chickens, turkeys, geese and the like) and mammals, such as primates, o vines, bo vines, porcines, ursines, caprines, felines, canines, equines, murines and the like, either grown in vivo or in vitro.
  • the nucleus is contained within a sperm head such as a membrane-challenged sperm head.
  • sperm head efficiently activates the injected oocyte sufficient for full development to term, following transfer of the developing embryo to a surrogate mother by a procedure known to those skilled in the art.
  • the sperm head may be treated by heat or another agent that ablates its ability to activate an oocyte, in which case oocytes are subjected to an activating stimulus following microinjection, to induce development.
  • Spermatozoa may be from amphibians, fish, birds (eg domestic chickens, turkeys, geese and the like) or mammals, such as primates, ovines, bovines, porcines, ursines, caprines, felines, canines, equines, murines and the like.
  • the method of the invention allows for a large range of injection pipette tip diameters.
  • Previous methods of transgenesis either do not permit the introduction of large segments of DNA (for example, where viral vectors are employed) or else may permit their introduction but with considerable technical difficulties.
  • the method of pronuclear microinjection is not suited to the high viscosity of preparations of artificial chromosomes; injection through the fine pipettes used (1-2 ⁇ m tip diameter) is difficult and DNA molecules often shear, resulting in failure.
  • handling is difficult, with such narrow tips tending to become more sticky after fewer uses.
  • pipettes used in the method of the invention now described are typically of tip diameter > 5 ⁇ m.
  • the relatively large tip diameter renders the handling of viscous DNA solutions easier, partly because needles are less sticky, and (ii) generates lower magnitude sheer forces; large DNA molecules are sensitive to damage by sheer.
  • a further advantage of the method of the invention is that it does not demand the injection of materials into a precise location within the oocyte. This is in contrast to pronuclear microinjection. This advantage is especially relevant to species whose oocyte cytoplasm is lipid-rich and thus opaque to light microscopy, such as the oocytes of many commercial species and breeds. Hence, the method of the invention does not require that the spatial relationship between the microinjection pipette tip and the oocyte cytoplasm be known precisely during microinjection.
  • tg NA is first mixed with components which stabilize the NA such as, but not limited to, basic proteins derived from sperm (eg protamines, perinuclear components and the like).
  • injection is into a cell which possesses a nucleus.
  • Such cells are exemplified by a met II oocyte (in which case the resulting embryo is a parthenogenote) or an enucleated met II oocyte into which a somatic or other nucleus had been implanted via nuclear transfer as described in Wakayama, T., Perry, A.C.F., Johnson, K., Zuccotti, M.
  • Oocytes are artificially stimulated to initiate development using a stimulus such as, but not limited to, Sr 2+ , ethanol or an electric pulse, according to known methods.
  • Microinjected cells are allowed to develop either following removal to suitable culture conditions in vitro, or to a suitable surrogate mother.
  • it is desirable to culture the cells to develop into an embryo and in a further embodiment of the invention, to examine embryos that had been cultured in vitro such that their development can be described and tg expression determined.
  • the method of the invention permits the selective transfer of transgenic embryos if they contain a tg whose expression can be monitored without killing the embryo. This is the case for GFP whose expression is driven in early embryos, as is the case for CMV-LE enhancer/chicken b-actin promoter combination. Expression may be monitored simply by viewing embryos briefly under long-wavelength (480 nm) ultra-violet UV illumination. Exposure to long-wavelength UV light is minimized to reduce potential UV damage to embryos, which damage may impair subsequent development of the embryo.
  • Oocyte donors B6D2F1
  • sperm donors B6D2F1
  • foster mothers ICR
  • Mature oocytes were collected from the oviducts of pregnant mare serum gonadotrophin- primed (5 IU), superovulated, 4-10-wk-old female B6D2F1 mice 14.5-16 h after the intraperitoneal administration of 5 IU human chorionic gonadotrophin (hCG).
  • the cumulus cell mass was dispersed by immediate treatment in CZB-H (CZB buffered with 20 mM HEPES, pH7.4; Chatot, C.L., Lewis, J.L., Torres I. & Ziomek, CA.
  • Piezo-electrically actuated microinjection of sperm heads into mouse eggs has been described by Kimura Y. & Yanagimachi, R. Biology of Reproduction 52, 709 (1995). Injections were usually completed within 18-19 h post hCG administration. The injection needle tip diameter was typically 5 ⁇ m. Test solution (usually with a single sperm head) was drawn into the pipette following expulsion of a small amount of mercury into the test solution droplet. This ensured that ahead of the mercury boundary, the test solution filled the pipette and was therefore not diluted further. The equivalent of approximately 1 pi was delivered into the ooplasm per microinjection.
  • Injected oocytes were maintained in operation medium (CZB-H) for approximately 2-10 min prior to transfer to CZB under mineral oil equilibrated in 5% (v/v in air) CO2 at 37»C.
  • oocytes were artificially activated by incubation, immediately after injection, for 45-60 min in Ca 2+ -free CZB containing 6.7 mM SrCl 2 , under mineral oil equilibrated in 5% (v/v in air) CO2 at 37°C. After this time, eggs were washed briefly in, and transferred to, fresh CZB, and incubation continued. Culture in vitro of embryos was in CZB for up to 4 days.
  • mice in 400 ⁇ l CZB medium Isolation of spermatozoa for triton X-100 extraction was by finely chopping two caudas epididymides at 0-l°C in Nuclear Isolation Medium (NIM: 125 mM KC1, 2.6 mM NaCl, 7.8 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , 3.0 mM EDTA; pH7.45) and
  • sperm suspension filtering the resulting sperm suspension to produce a final volume of 900 ⁇ l.
  • Piezo-actuated microinjection of oocytes and culture of embryos in CZB under mineral oil equilibrated in 5% (v/v in air) CO2 at 37°C has been detailed elsewhere.
  • sperm heads were aspirated into a pipette attached to a piezo electric pipette-driving unit and one injected per oocyte. Oocytes that lysed soon after injection were discarded. Where appropriate, dislocation of heads from tails was by the application of a single piezo pulse to the region of the sperm mid-piece.
  • EXAMPLE 2 Exposure of sperm nuclei to GFP or ⁇ -galactosidase tg NA by mixing: production of transgenic embryos
  • the large (3.5 kb) SalGl-BamHL fragment of plasmid pCX-EGFP used here harbors a GFP gene expressed from a strong CMV-LE chicken ⁇ -actin enhancer-promoter combination (Niwa, H., Yamamura, K. & Miyazaki, J. Gene 108, 193 [1991]), but lacks a eukaryotic origin of replication (Zhang, G. Vanessa, G. & Kain, S.R. Biochemical and Biophysical Research Communications 227, 707 [1996]; Takada, T. Iida, K. Awaji, T. Itoh, K. Takahashi, R. Shibui, A. Yoshida, K.
  • triton X-100 extraction 100 ⁇ l 0.5% (v/v in NIM) triton X-100 was added to 900 ⁇ l sperm suspension in NIM (see example 1) and mixed by trituration for 30 sec on ice. Cells were pelleted by centrifugation for 1 min at 20,000g, 2°C and thoroughly resuspended in 2 ml ice-cold NLM before repelleting for 2 min at 20,000g, 2°C. This final pellet was resuspended in 400 ⁇ l
  • Embryos were examined 3 - 3.5 days after microinjection by epifluorescence microscopy for expression of GFP using a UV light source (480 nm) with FITC filters. This enabled the clear identification of non-fluorescent (te non-GFP-expressing), weakly-fluorescent and strongly-fluorescent embryos and mosaic embryos (containing both fluorescent and non- fluorescent cells), which were scored accordingly.
  • indolyl- ⁇ -D-galactopyranoside X-gal. Embryos were examined and scored by light microscopy. Results, presented in Table 1 below, demonstrate that the method of the invention generates transgenic embryos with a high efficiency. The method yielded detectably tg expressing embryos when the pCX-EGFP DNA fragment concentration was 500 pg/ ⁇ l but not 50 pg/ ⁇ l. This suggests that the method is sensitive at average DNA concentrations corresponding to as few as approximately 15-150 molecules per injection.
  • Fragment 1 - treatment No. oocytes m-b (%) * - ⁇ +/- ⁇ + * ⁇
  • +Exogenous DNA fragments were pCX-EGFP- ⁇ mffl-S ⁇ GI or pxCANLacZ-S ⁇ /GI, SalG -Xh ⁇ l, or Xhol. Fragments were mixed with sperm heads at DNA concentrations of 5-10 ng/ « 1. With the exception of the last three rows (see Example X), exogenous DNA was injected after mixing with sperm samples as described in Example 2.
  • ⁇ Tg expression -, negative; +, positive; +/-, m-b containing both + and - cells (mosaics).
  • Single-shot double transgenesis was used to generate embryos co-expressing two tgs after a single microinjection as described in Example 1, with the following modifications.
  • Sperm heads were co-injected with a DNA solution containing: 2.5 ng/ ⁇ l pCX-EGFP SalGl-BamHl
  • pCX-LacZ is a derivative of pCX-
  • embryos were first scored for GFP expression and then for ⁇ -galactosidase expression as described in examples 1 and 2 respectively.
  • embryos were mounted between a microscope slide and cover slip and images collected to show development and GFP expression, prior to fixation and staining to show LacZ expression.
  • the sperm suspension in each washing experiment was divided into two 5 ⁇ l aliquots immediately after mixing and incubating with pCX-EGFP DNA for 1 min.
  • One aliquot (washed sperm) was diluted and washed by mixing well with 50 ⁇ l ice-cold, fresh CZB or NLM. Both aliquots were then pelleted for 2 min at 20,000g, 2°C.
  • the supernatant from the washed sperm aliquot was carefully removed and replaced with 5 ⁇ l fresh CZB or NIM; the supernatant from the second aliquot was used to resuspend its own pellet (this sample was therefore not washed).
  • Results, presented in Table 1, are strongly suggestive of the ability of a nucleus and NA to associate in vitro prior to microinjection.
  • sperm heads that had been subjected to one of the three membrane disruption procedures were co-injected with pCX-EGFP DNA, the resulting embryos were cultured in vttr ⁇ for up to approximately 3.5 days (to the morula-blastocyst stage) and embryos were then transferred to surrogate mothers non-selectively (ie not on the basis of fluorescence).
  • Phenotypic analysis of tg integration was by examination of offspring under long-wave UV. A high proportion (17 - 21%) of offspring were transgenic with respect to observable GFP expression in skin (Table 2 Below); this efficiency did not depend on the membrane disruption method used to prepare spermatozoa. Rates of zygotic development to term were comparable for each of the three groups of membrane-disrupted sperm heads (12 - 14%).
  • Triton X-100 218 150 (9; 31 6 a
  • Hm-b morulae-blastocysts. Values in parentheses show the number of surrogate mothers used as recipients in embryo transfers
  • ⁇ Tg expression + , positive pups are those expressing GFP ectopically in their skin.
  • Tail-tip biopsies from 3 to 6 week-old, randomly-selected green pups and their non-green litter-mates were used for extraction of total, genomic DNA.
  • Photography of tails was under a fluorescent stereomicroscope equipped with a 480/40 nm filter.
  • 10 ⁇ g genomic DNA per sample was digested with EcoRI and probed with the 733bp EcoRI fragment of pCX- ⁇ GFP.
  • Oligonucleotide primers used for the detection of the GFP gene by PCR of 1 ⁇ g genomic DNA per reaction were forward (TTGAATTCGCCACCATGGTGAGC) and reverse
  • reaction parameters were 95°C for 9 min (1 cycle); 94°C for 45 sec, 60°C for 30 sec, 72°C for 45 sec (40 cycles). ⁇ lectrophoretically separated products were visualized following ethidium bromide staining.
  • tg vectors that contain an internal ribosome entry site (LRES).
  • LRES internal ribosome entry site
  • Embryos are generated by microinjection as described in Example 1.
  • the DNA construct contains the GFP gene expressed from a strong CMV-LE chicken ⁇ -actin enhancer- promoter combination of Example 2, flanked by lox sites. This enhancer-promoter is adjacent to a second element sufficient to drive the programmed expression of any given tg.
  • the GFP open reading frame is adjacent to the given tg open reading frame, separated by an IRES.
  • the tg DNA construct is mixed with demembranated spermatozoa and coinjected into the met II oocyte of a strain expressing the Cre recombinase under the control of, for example the goosecoid gene promoter, which functions at the gastrulation stage of development, thereby excising the CMV-LE/chicken ⁇ -actin enhancer-promoter element during that stage.
  • Embryos are cultured in vitro for up to approximately 3.5 days (to the morula-blastocyst stage) and transferred to surrogate mothers selectively (ie on the basis of fluorescence) to enable their full development.

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Abstract

L'invention concerne un procédé de production d'animaux et de cellules transgéniques par la co-insertion d'acide nucléique et d'un noyau dans un ovocyte non fécondé. De préférence, la co-insertion est effectuée par micro-injection, et idéalement par micro-injection piézo-électrique. Des embryons exprimant le transgène (tg) sont produits après co-injection d'ovocytes de souris non fécondés à l'aide de têtes de sperme et d'ADN exogène codant pour une protéine fluorescente verte (GFP) ou pour un rapporteur de galactosidase β. On peut laisser se développer l'ovocyte micro-injecté en cellules différenciées ou en cellules souches ; en embryon in vitro avant transfert dans une mère porteuse hôte ; ou on peut le transférer directement dans une mère porteuse hôte. Le développement embryonnaire peut se faire à terme, de sorte que la progéniture acquiert des modifications transgéniques pouvant modifier ses caractéristiques (phénotype), et qui peuvent ensuite être transmises à la descendance.
EP99942164A 1998-08-11 1999-08-11 Procede de mise en oeuvre de transgenese Withdrawn EP1111991A4 (fr)

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WO2000009674A1 (fr) 1998-08-11 2000-02-24 University Of Hawaii Transgenese mammifere par injection de sperme intracytoplasmique
EP1241935A2 (fr) * 1999-12-17 2002-09-25 Gerald Schatten Procedes de production d'animaux transgeniques
US7067308B1 (en) * 2000-03-28 2006-06-27 Bioagri Corporation Vector for genetically modifying non-human animals
US7053187B2 (en) 2000-03-28 2006-05-30 Gioagri Corporation Sperm-specific monoclonal antibody, mAbC
US6846306B1 (en) * 2000-10-10 2005-01-25 Cold Spring Harbor Laboratory Single cell electroporation
CN101407807B (zh) * 2008-11-19 2013-11-06 暨南大学 对染色体进行处理而改变细胞遗传特性的方法及其应用
FR2969497B1 (fr) * 2010-12-27 2013-06-28 Ceva Sante Animale Composition luminescente comme biomarqueur dans un oeuf aviaire, dispositif et procede correspondants.
CN104372016A (zh) * 2013-10-19 2015-02-25 刘立新 在受体细胞的目的基因组序列引入变异的系统和方法
CN104911212A (zh) * 2015-05-27 2015-09-16 中国科学院广州生物医药与健康研究院 一种高效转染HaCaT细胞的方法

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