WO2002019811A2 - Production d'animaux transgeniques par transfert nucleaire et par emploi d'oocytes au stade de vesicule germinative - Google Patents

Production d'animaux transgeniques par transfert nucleaire et par emploi d'oocytes au stade de vesicule germinative Download PDF

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Publication number
WO2002019811A2
WO2002019811A2 PCT/CA2001/001305 CA0101305W WO0219811A2 WO 2002019811 A2 WO2002019811 A2 WO 2002019811A2 CA 0101305 W CA0101305 W CA 0101305W WO 0219811 A2 WO0219811 A2 WO 0219811A2
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oocytes
oocyte
somatic cell
derived
pronucleus
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PCT/CA2001/001305
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English (en)
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WO2002019811A3 (fr
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Bin Wang
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Nexia Biotechnologies, Inc.
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Priority to AU2001291558A priority Critical patent/AU2001291558A1/en
Publication of WO2002019811A2 publication Critical patent/WO2002019811A2/fr
Publication of WO2002019811A3 publication Critical patent/WO2002019811A3/fr

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    • 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
    • C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos

Definitions

  • This invention relates to methods for the generation of transgenic animals.
  • Completion of the meiotic cell cycle requires (1) oocyte growth, along with proliferation of the surrounding somatic cells, until the oocyte is fully grown; and (2) exposure to a preovulatory gonadotropin surge, which occurs cyclically after puberty.
  • oocytes develop their competence to resume meiotic maturation, but remain arrested at the GV stage within follicles, until those destined to ovulate respond to gonadotropin stimulation (Yanagimachi, "Mammalian Fertilization," The Physiology of Reproduction, eds. E. Knobil, J. Neill, L.L. Ewing, G.S. Greenwald, C.L. Markert, and D.W.
  • the present invention employs a GV stage oocyte as the recipient of a somatic cell for nuclear transfer.
  • the somatic cell is genetically modified.
  • the reconstructed GV oocyte is then matured by in vitro culture, generating an Mil stage oocyte whose haploid genome is derived from the introduced genetically modified somatic cell nucleus.
  • the oocyte is then activated and the somatic cell pronucleus is removed and fused into a pronuclear stage embryo (zygote) with one of its two pronuclei already removed.
  • the reconstructed zygote is next cultured and transferred to a recipient mother to develop to term.
  • the invention features a method for generating a non-human transgenic animal containing a desired gene.
  • the method involves recovering a germinal vesicle stage oocyte from a donor animal; providing a somatic cell containing the desired gene; fusing the somatic cell with the oocyte to form a fused couplet; allowing the couplet to mature; and activating it to produce an activated couplet containing an oocyte-derived pronucleus and a somatic cell-derived pronucleus.
  • the somatic cell-derived pronucleus is then removed from the activated couplet and fused with a pronucleus stage embryo lacking one pronucleus, to form a reconstructed zygote.
  • This reconstructed zygote, or a cleaved embryo, morulae, or blastocyst formed from culturing the zygote, is then transferred into a recipient animal, and developed to term to produce a transgenic animal containing a desired gene.
  • the germinal vesicle stage oocyte contains a nucleus or is enucleated.
  • the somatic cell may be in G ls G 2 , or M phase of the cell cycle.
  • the animal is preferably a mammal, more preferably a ruminant, for example, a sheep, a goat, or a cow, or it may be a pig or a rabbit.
  • the somatic cell and germinal vesicle stage oocyte can be derived from the same animal, or from different animals, and may be from the same or different species.
  • the couplet can be activated by parthenogenetic activation or by sperm-mediated activation.
  • transgene is meant any piece of nucleic acid that is inserted by artifice into a cell, or an ancestor thereof, and that becomes part of the genome of the animal which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic animal, or may be a gene homologous to an endogenous gene of the animal.
  • transgenic is meant any cell which includes a nucleic acid sequence that has been inserted by artifice into a cell, or an ancestor thereof, and that becomes part of the genome of the animal which develops from that cell.
  • the transgenic animal is a transgenic mammal (e.g., a rodent or ruminant).
  • the nucleic acid (transgene) is preferably inserted by artifice into the nuclear genome.
  • a “germinal vesicle stage oocyte” is meant an oocyte whose nucleus is arrested in a postsynaptic stage of meiotic prophase I.
  • a "somatic cell” is meant any cell that does not contribute to the production of a gamete.
  • a "cleaved embryo” is meant a zygote that has undergone a number of cell divisions and is in the process of developing into a morula or a blastocyst.
  • an “elite animal” is meant an animal that is highly valuable in terms of genetic traits in productivity (for example, milk yield or protein production), reproduction, or disease resistance.
  • An elite animal may be an animal that contains a foreign gene encoding a protein of commercial value.
  • An elite animal may also be an endangered species.
  • the methods of the invention are more efficient than standard microinjection and animal cloning by nuclear transfer techniques.
  • the invention provides superior elite animal reproduction for the following reasons.
  • somatic cell nucleus goes through oocyte maturation, fertilization, activation, and pronuclear stage and early embryo development, the donor cell genome has more chances to be completely reprogrammed.
  • somatic cell nucleus is introduced into the oocyte later, for example, after the oocyte has matured.
  • the introduction of a somatic cell nucleus into a GV stage oocyte will lead to higher survival rates of the embryos and healthier offspring after embryo transfer.
  • oocyte activation is carried out by an intact sperm, the development of the reconstructed embryos will be superior to that from artificial activation used in other animal cloning by nuclear transfer techniques.
  • the donor cell's genome can be modified through standard procedures, so that a foreign gene can be introduced into the offspring developed from the reconstructed oocytes. If the foreign gene integration site in the donor cell is single, the transgenic rate should be 50% of the offspring, based on the fact that a foreign gene has a fifty percent chance of being expelled into the first polar body. If the integration sites are multiple, the transgenic rate will be 100%.
  • the animal's genome can be precisely modified through gene target technology, which cannot be achieved by traditional microinjection methods.
  • FIG. 1 is a schematic representation of the steps involved in producing a transgenic embryo through GV oocyte reconstruction according to the invention.
  • GV stage oocytes are stripped of cumulus cells.
  • a somatic cell carrying the foreign gene is introduced into the perivitelline space at the opposite position of the germinal vesicle.
  • the oocytes are subjected to artificial parthenogenetic activation through a procedure that could lead to second polar body release (if donor cells are synchronized at G 2 or M phase during nuclear transfer) or no second polar body release (if donor cells are at G 0 or Gi phase).
  • the somatic cell-derived pronucleus is then separated from the reconstructed oocyte based on its pronuclear location in the cytoplasm; the pronucleus near the slit on the zona pellucida, which is created during cell transfer, is presumed to be of somatic cell origin.
  • the separated pronucleus is transferred and fused into a pronucleus stage embryo whose female or male pronucleus has been removed previously (depicted on the right side of the schematic).
  • This reconstructed zygote is cultured in vitro to develop to a suitable embryo stage, and transferred into a foster mother for further development.
  • Bovine ovaries were collected from a local abattoir and transported to the laboratory in a thermos containing D-PBS (Gibco, Grand Island, NY) at 20-25°C. Within 1-2 hours after ovary arrival, the oocytes (in the form of cumulus-oocyte complexes) were aspirated from 2- to 5-mm ovary follicles, and cultured in maturation medium (TCM199 (Gibco) supplemented with 10% heat inactivated fetal calf serum (Immunocorp), 0.1% gentamicin (Gibco), 5 ⁇ g/ml luteinizing hormone (Noble Laboratory), 0.5 ⁇ g/ml follicle-stimulating hormone (Noble Laboratory), 1 ⁇ g/ml estradiol (Sigma), and 22 ⁇ g/ml pyruvate (Gibco)).
  • TCM199 maturation medium
  • Immunocorp gentamicin
  • Noble Laboratory 0.5 ⁇ g/
  • the culture environment was 5% C0 2 and 95% humidified air at 38.5°C.
  • the cumulus cells were removed to denude the oocytes, by vortexing the cumulus-oocyte complex for 1.5 minutes.
  • the germinal vesicle (GV) in each oocyte was located under a Nomarski optic 400X field.
  • a 20-25 ⁇ m slit was made on the zona pellucida near the GV.
  • a small amount of cytoplasm along with the GV was squeezed out of the slit by pushing the oocytes with a microglass needle.
  • the karyoplasts were pinched off by pipetting the oocytes through capillary glass tubing, producing enucleated oocytes.
  • the enucleated oocytes were temporarily cultured in droplets of Emcare embryo flushing media containing 1% FCS on a 37°C warm plate, until transfer of the somatic donor cell.
  • a bovine fetal fibroblast cell line was maintained in culture using standard cell culture methods. On the day of somatic cell transfer, the fibroblast monolayer was trypsinized and resuspended in Emcare containing 1% FCS. This cell suspension solution was transferred into the perivitelline space (PVS) of enucleated oocytes or non-enucleated GV oocytes at the opposite position of the GV, through microinjection with a 15 ⁇ m beveled glass pipette. The cells were gently pushed against the oocyte membrane in order to make tight contact between the cell membranes.
  • PVS perivitelline space
  • somatic cell and oocyte couplets were then cultured in micromanipulation drops of maturation medium (TCM199 media containing 25 mM HEPES supplemented with 10% FCS, 0.01 units/ml bFSH, 0.01 units/ml bLH, 1 ⁇ g/ml E 2 (estradiol), 22 ⁇ g/ml sodium pyruvate, and 1 ⁇ g/ml gentamicin) until electrofusion.
  • TCM199 media containing 25 mM HEPES supplemented with 10% FCS, 0.01 units/ml bFSH, 0.01 units/ml bLH, 1 ⁇ g/ml E 2 (estradiol), 22 ⁇ g/ml sodium pyruvate, and 1 ⁇ g/ml gentamicin
  • TCM199 media containing 25 mM HEPES supplemented with 10% FCS, 0.01 units/ml bFSH, 0.01 units/ml bLH, 1 ⁇ g/m
  • the electrofusion medium was 0.25 M D-Sorbital (Sigma) containing 100 ⁇ M CaOAc (Sigma), 0.5 mM MgOAc (Sigma), and 0.1% BSA (Fatty acid free, Sigma). Fusion pulse parameters were maintained at a constant single 1.84 KV/CM pulse for 15 ⁇ sec. The time that the electrofusion occurred varied from one experiment to another, ranging from 4 to 7 hours post-oocyte culture. Fused couplets were separated from non-fused couplets and cultured in maturation medium, as described above for 16 to 22 hours to mature the oocytes.
  • bovine zygotes were derived by in vitro maturation and in vitro fertilization. The procedure is briefly described as follows:
  • the oocytes were aspirated from 2-5 mm size follicles from the ovaries obtained from a local slaughter house.
  • the oocytes with the intact cumulus cell mass and homogenous cytoplasm were selected for in vitro maturation.
  • the maturation medium was described as above.
  • oocytes were inseminated by freeze-thawed bull spermatozoa.
  • zygotes were stripped of cumulus cells and centrifuged at 15,600 g for 6 minutes to stratify the cytoplasm for visualization of pronuclei.
  • the pronuclear stage embryos from the NT procedure and the recipient zygotes were centrifuged at 15,600 g for 6 minutes in order to visualize the pronuclei under Nomarski contrast optics.
  • a 45 ⁇ m beveled pipette was inserted into the PVS of the NT-derived oocyte to aspirate the pronucleus near the slit on the zona pellucida.
  • the somatic cells are at G 0 or Gi during GV oocyte reconstruction, the DNA content of the somatic cell nucleus is only 50% that of the germinal vesicle-originated pronucleus. After activation, the size of the somatic cell-derived pronucleus should be smaller than the germinal vesicle-originated pronucleus germinal vesicle.
  • the donor pronucleus was then transferred into the PVS of the pronuclear recipient zygote whose female pronucleus was previously removed.
  • the pronuclear exchange was completed using the same electrofusion parameters described above.
  • the reconstructed zygotes were cultured in S OF medium at 38.5°C under 5% C0 2 , 7% 0 2 , and 88% N 2 for 7 days to examine its in vitro development.
  • Triton X 100 solution and stained with Hoechst 33342. The samples were mounted on glass slides, and the nuclei and chromatin were evaluated under an epifluorescence microscope.
  • Metaphase oocytes were first removed of zona pellucida by digestion in a solution containing Emcare and 0.2% pronase E (from streptomyces griseus, Sigma), followed by hypotonic treatment in 40% fetal calf serum in deionized water for 7 minutes. The oocytes were then fixed with acetic acid, methanol, and *
  • the GV stage oocytes were denuded and enucleated, as described above.
  • the somatic bovine fibroblast cells were transferred and fused into the cytoplasm of each ova, also as described above.
  • the cell cycle of the donor cell was not synchronized in this experiment.
  • Approximately 16 and 22 hours after oocyte isolation, the reconstructed oocytes were stained and evaluated for nuclear maturation. This was done by staining the oocytes with Hoechst 33342, and observing the morphology of the chromatin using an epifluorescence microscope.
  • the nucleus can be readily categorized to be GV, MI, Mil, or interphase between MI and MIL
  • denuded oocytes were cultured in IVM medium under the same conditions as the reconstructed oocytes. The experiment was replicated three times. The results from this study are shown in Table 1. A high GV enucleation rate (95%) was obtained in this study. When the reconstructed oocytes were cultured for 16 hours, 100% of the transferred somatic cell nuclei became condensed; by 22 hours, the majority of oocyte nuclei (71%) still remained condensed, with only a few reaching the MI, MI-MII, or Mil stage.
  • the GV stage oocytes were denuded, as described above, but they were not enucleated.
  • a bovine fibroblast cell was transferred and fused into the cytoplasm of each ovum, also as described above. Again, the cell cycle of the donor cells was not synchronized.
  • the level of maturation was compared to that of denuded oocytes cultured in IVM medium under the same conditions as the reconstructed oocytes. At 16 and 22 hours of maturation culture after oocyte isolation, the reconstructed oocytes were evaluated for their level of maturation, as described above. The experiment was replicated three times.
  • NT nuclear transfer
  • PBs polar bodies
  • Non-enucleated bovine cumulus cells and fetal fibroblast cells were starved for 3-4 days to synchronize the cell cycles at the Gr/Gi stage. Chromatin condensation in the nuclei of cells at Gr Gi phase can produce chromosomes with a single chromatid. Therefore, in the Mil stage oocytes derived from GV-NT, the chromosomes from somatic cells can be distinguished from chromosomes of the oocyte itself. Mature oocytes and the Mil oocytes fused with Go/Gi cells were used as controls to evaluate the chromosome morphology. The experiment was replicated 3 times.
  • NT nuclear transfer
  • PB ⁇ olar body
  • the reconstructed oocytes were activated by either spermatozoa, through standard IVF procedures, or by artificial activation with calcium ionophore combined with 6-DMAP.
  • non-enucleated GV oocytes were designated for parthenogenetic activation by in vitro fertilization using spermatozoa. At about 18 hours post-insemination, the oocytes were stained to evaluate the pronuclear development. Three replicates of this experiment were conducted.
  • the competence of the somatic cell nucleus developing into a pronucleus could not be examined accurately using standard IVF procedures. Therefore, artificial activation was carried out using A23187 and 6-DMAP. Briefly, the oocytes were first treated with A23187 for 5 minutes and then treated with 6-DMAP for 4 hours or overnight. Treatment with A23187 is done to increase the calcium in the cytoplasm, which is well recognized as a signal to activate the cell cycle. 6- DMAP is used to inhibit the activity of maturation promoting factor (MPF), leading to full activation of an oocyte. The oocytes with two first polar bodies were chosen for activation treatment after 22 hours of maturation culture. The development of the nuclei into pronuclei was then measured 18 hours post- activation.
  • MPF maturation promoting factor
  • the competence of the somatic cell-derived pronucleus was further investigated.
  • a female pronucleus in the fertilized recipient oocyte was replaced by a somatic cell- derived pronucleus, and the reconstructed zygotes were cultured in Gl + G2 medium for 7 days.
  • the zygotes generated by pronuclear exchange within fertilized oocytes were used as controls.
  • the results of this study are given in Table 7.
  • the initial cleavage rate of GV-NT-derived oocytes was similar to the control (74% for the GV-NT-derived oocytes versus 80% for the control oocytes).
  • a small proportion of zygotes (4%) developed to the blastocyst stage in the GV- NT group.
  • the pronucleus exchange micromanipulation procedure does not appear to be harmful to embryo development, since the NT control group had a 50% blastocyst development rate.

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Abstract

Cette invention concerne des méthodes de production d'animaux transgéniques au moyen de techniques de transfert nucléaire et d'oocytes au stade de vésicule germinative.
PCT/CA2001/001305 2000-09-06 2001-09-06 Production d'animaux transgeniques par transfert nucleaire et par emploi d'oocytes au stade de vesicule germinative WO2002019811A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1958964A2 (fr) 2004-02-24 2008-08-20 The Government of the United States of America, as represented by The Secretary, Department of Health and Human Services RAB9A, RAB11A, et modulateurs correspondants liés à une maladie infectieuse
WO2009001224A2 (fr) 2007-06-22 2008-12-31 Eth Zurich Antiviraux
EP2311530A2 (fr) 2004-10-27 2011-04-20 Vanderbilt University Genes mammaliens intervenant dans une infection
US10202615B2 (en) 2010-12-10 2019-02-12 Vanderbilt University Mammalian genes involved in toxicity and infection

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau
WO1999037143A2 (fr) * 1998-01-21 1999-07-29 University Of Hawaii Developpement a terme d'animaux a partir d'ovocytes enuclees reconstitues avec des noyaux de cellules somatiques adultes
WO2000025578A2 (fr) * 1999-04-26 2000-05-11 Trustees Of Tufts College Procedes de clonage des animaux
WO2000042174A1 (fr) * 1999-01-13 2000-07-20 Ppl Therapeutics (Scotland) Limited Procede de double transfert de noyau et resultats de ce transfert

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997007669A1 (fr) * 1995-08-31 1997-03-06 Roslin Institute (Edinburgh) Populations de cellules quiescentes pour transfert de noyau
WO1999037143A2 (fr) * 1998-01-21 1999-07-29 University Of Hawaii Developpement a terme d'animaux a partir d'ovocytes enuclees reconstitues avec des noyaux de cellules somatiques adultes
WO2000042174A1 (fr) * 1999-01-13 2000-07-20 Ppl Therapeutics (Scotland) Limited Procede de double transfert de noyau et resultats de ce transfert
WO2000025578A2 (fr) * 1999-04-26 2000-05-11 Trustees Of Tufts College Procedes de clonage des animaux

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Title
DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 11 December 2000 (2000-12-11) GRABAREK JOANNA ET AL: "Oocyt jajnikowy: Nowy typ komorki-biorcy w klonowaniu ssakow? ("Germinal vesicle oocyte: A new type of recipient cell in cloning of mammals?")" Database accession no. PREV200100331925 XP001122811 -& PRACE I MATERIALY ZOOTECHNICZNE, no. 57, 2000, pages 103-110, XP001122811 ISSN: 0137-1649 & GRABAREK J., 24 February 2000, Pzeksztalcanie jader blastomerow myszy w enukleowanych oocytah w stadium pecherzyka zarodkowego. Rozprawa doktorska, IGHZ PAN w Jastrzebcu. *
OGURA ATSUO ET AL: "Development of normal mice from metaphase I oocytes fertilized with primary spermatocytes." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 95, no. 10, 12 May 1998 (1998-05-12), pages 5611-5615, XP002118958 May 12, 1998 ISSN: 0027-8424 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1958964A2 (fr) 2004-02-24 2008-08-20 The Government of the United States of America, as represented by The Secretary, Department of Health and Human Services RAB9A, RAB11A, et modulateurs correspondants liés à une maladie infectieuse
EP2311530A2 (fr) 2004-10-27 2011-04-20 Vanderbilt University Genes mammaliens intervenant dans une infection
WO2009001224A2 (fr) 2007-06-22 2008-12-31 Eth Zurich Antiviraux
US10202615B2 (en) 2010-12-10 2019-02-12 Vanderbilt University Mammalian genes involved in toxicity and infection

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WO2002019811A3 (fr) 2003-01-16

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