EP1589811A2 - Microperforation sans contact de zona pellucida d'ovocytes avec un faisceau laser a diode de 1,48 m pour l'introduction de retrovirus - Google Patents

Microperforation sans contact de zona pellucida d'ovocytes avec un faisceau laser a diode de 1,48 m pour l'introduction de retrovirus

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Publication number
EP1589811A2
EP1589811A2 EP04708744A EP04708744A EP1589811A2 EP 1589811 A2 EP1589811 A2 EP 1589811A2 EP 04708744 A EP04708744 A EP 04708744A EP 04708744 A EP04708744 A EP 04708744A EP 1589811 A2 EP1589811 A2 EP 1589811A2
Authority
EP
European Patent Office
Prior art keywords
laser
zona pellucida
embryo
retrovirus
laser beam
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.)
Withdrawn
Application number
EP04708744A
Other languages
German (de)
English (en)
Inventor
Alfred Senn
Thierry Pedrazzini
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.)
Octax Microscience GmbH
Vitrolife GmbH
Original Assignee
MTG Medical Technology Vertriebs GmbH
Octax Microscience GmbH
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 MTG Medical Technology Vertriebs GmbH, Octax Microscience GmbH filed Critical MTG Medical Technology Vertriebs GmbH
Publication of EP1589811A2 publication Critical patent/EP1589811A2/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • lentiviruses are a class of retroviruses that cause chronic illnesses in the host organisms they infect .
  • retroviruses lentiviruses have the distinguishing property of being able to infect both dividing and nondividing cells, and this ability has led to their development as gene delivery vehicles. Because of their property to infect both dividing and non-dividing cells, they have been used widely as gene delivery tools.
  • lentiviruses engineered to carry a transgene were injected into the perivitelline space of single cell mouse embryos. Embryos were implanted into pseudo-pregnant mothers, and carried to term.
  • Embryos could be maintained in their usual culture dish and medium during the drilling process without requiring special quartz optical equipment as for UV lasers (Sch ⁇ tze et al . , Fertil . Steril . , 61, 783-786, 1994), a change of medium (Blanchet et al . , Fertil. Steril., 57, 1337-1341, 1992) or micromanipulators as for the 2.9 ⁇ m Erbium:YAG laser (Obruca et al . , Hum. Reprod.
  • a need remains to provide a procedure to produce transgenic animals at a high rate. Specifically, a need remains to provide a method without an injection and without the necessity for long-term culture of embryos.
  • the above object is overcome by the present invention by the use of a 1.48 ⁇ m laser to microdrill a hole in the zona pellucida of a single-cell embryo to introduce a retrovirus, preferably a lentivirus" carrying a particular transgene into said embryo.
  • the present inventors used the above method to allow the retroviral vectors to be in contact with the plasma membrane of the embryos.
  • Drilled fertilized oocytes were incubated for 2-24 hours preferably, for 3 to 5 , more preferred 4 hours with lentiviruses carrying a particular transgene. Because of the partial denudation, this makes the physical injection of the viral particles under the zona pellucida unnecessary.
  • incubation time can be decreased to a few hours.
  • the presence of the zona pellucida around the embryos make them stronger and more resistant to the whole procedure.
  • the embryo can be transferred back into foster mothers the same day, and therefore the technique is less time consuming.
  • the hole as microdrilled in the zona pellucida of a single-cell embryo is between 2 to 60 ⁇ m, preferably between 2 to 20 ⁇ m in diameter, more preferably 5 to 15 ⁇ m, even more preferred 5 - lO ⁇ m.
  • the zona pellucida of the single-cell embryo is exposed to one or up to 10 shots, preferably 1 to 5 shots, more preferred once or twice to 0.1 ⁇ s to 100 ms, preferably 0.1 to 15 ms of laser light, preferably 0.5 to 10 ms of laser light, more preferably 1 to 5 ms of laser light, depending on oocyte species and for laser power in focus of 20 - 500 mW, preferably 20 - 250 mW, more preferred 100 - 160 mW.
  • the laser can be provided at any point of an optical system.
  • Said optical system can in an exemplary embodiment be a microscope.
  • the laser is mounted directly in front of the objective (as seen from viewer to object) ; in another embodiment the laser is provided in front of or after the eye-piece or can replace e.g. the eye-piece or the CCD camera.
  • the laser could in a further embodiment also be provided within the objective, e.g. in front of the last one to six lenses, preferably two to four lenses, of the objective.
  • the actual arrangement of the laser is not restricted to the above embodiments.
  • the laser can be provided together with any optical system, e.g. a microscope, in a preferred embodiment an inverse microscope.
  • Fig. 1 The 1.48 ⁇ m diode laser assisted hatching unit.
  • Fig. 1A The FERTILASE® system, with its control unit on the right, is attached to the fluorescence port at the back of the inverted microscope.
  • Fig. IB The compact OCTAXTM system, with its octagonal laser in the back and the miniaturised video camera on the left.
  • Fig. 2 Schematic of the 1,48 ⁇ m diode zona pellucida drilling arrangement.
  • the fluorescence port of the inverted microscope is used to couple the surgical laser.
  • the microscope objective is used to precisely focus the laser radiation onto the egg ZP.
  • Fig. 3 Zona pellucida drilling strategy.
  • the focused laser beam is directed tangentially to the ZP to produce a trench.
  • Fig. 4 Human zygote drilled at the 2-pn stage on day 1.
  • the laser drilled trench opens completely the ZP; it has been obtained by two consecutive 9 ms irradiation with the OCTAX laser system.
  • Fig. 5 Electron micrograph showing a trench drilled with the 1.48 ⁇ m diode laser system in a mouse zygote. Note the sharpness of the walls of the opening.
  • a 1.48 ⁇ m diode laser is used, preferably one of the type that was developed by the Institut d'Optique Appliquee (K. Rink and G. Delacretaz; at the Lausanne, Switzerland) in association with the Reproductive Medicine Unit (DGO; CHUV) (Rink et al . 1994 Supra; Rink et al . 1996 Supra) and commercialized as a functional unit (Fertilase ⁇ ; formerly Medical Technologies Montreux S.A., Clarens, Switzerland, now OCTAX) .
  • DGO Reproductive Medicine Unit
  • CHUV Reproductive Medicine Unit
  • Fertilase ⁇ formerly Medical Technologies Montreux S.A., Clarens, Switzerland, now OCTAX
  • ZP Zona pellucida Opening is performed according to the following procedure.
  • the culture dish is placed on the displacement stage of the microscope ( Figure 2) .
  • Opening is performed by exposing the ZP to the laser beam during 0.1 - 15 ms, preferably during 0.5-10 ms, more preferred 1-5 ms .
  • the hole size can be chosen by varying the ' irradiation time, typically a hole having a diameter of 20 ⁇ m is produced with a 12-30 ms irradiation time. Larger hole diameters are obtained by increasing the irradiation time. If the egg is placed tangentially to the diode laser beam a trench is induced in the ZP. By precisely positioning the laser focalization point with respect to the ZP width a complete opening or only a local thinning of the ZP can be generated at will ( Figure 3) .
  • the above technique can be used for all single-cell embryos comprising a zona pellucida. Particularly, it can be used for all mammals, including human and non-human embryos. In a more preferred embodiment, the embryo is a mouse or rat embryo .
  • the lentiviral backbone which can be used in these methods is based on a self-activating vector described previously (H. Miyoshi et al . , L.Virol. 72, 8750, (1998).
  • Plasmid pFUGW is based on the HRCS-G vector gift of I. Verma, Salk Insitute, La Jolla, CA) constructed by inserting into its multicloning site the HIV-1 flap sequence polymerase chain reaction (PCR) -amplified from the HIV NLAA3 genome, the human polyubiquitin promoter C (gift of L. Thiel , Amgen, Thousand Oaks, CA) , the* GFP gene and the WRE (gift of D. Trono, University of Geneva, Geneva, Switzerland) .
  • PCR HIV-1 flap sequence polymerase chain reaction
  • Lentiviral vectors were produced by co-transfecting the transfer vector of pfUGW, the HIV-1 packaging vector ⁇ 8.9 and the VSVG envelope glycoprotein into 293 fibroblasts and concentrated as described previously, FUGW viruses were titered on 293 fibroblasts. Serial dilutions of the virus were applied to the cells, and infectivity was determined after 72 hours by fluorescence microscopy for GPF expression.
  • the vector was engineered to carry an internal promoter driving the GFP reporter gene.
  • the human ubiquitin-C promoter was found to provide the most reliable expression across different cell types and was selected for subsequent experiments .
  • the wood-chuck hepatitis virus posttranscriptional regulatory element (WRE) was inserted downstream of GFP.
  • WRE wood-chuck hepatitis virus posttranscriptional regulatory element
  • the human immunodeficiency virus-I (HIV-1) flap element V. Zennon et al . , Cell 101, 173, (2000)
  • LTR 5 ' long terminal repeat
  • Viruses were pseudotyped with the vesicular stomatitis virus glycoprotein (VSVG) and concentrated by ultra-centrifugation to approximately 1x10 s infectious units (I.U.)/ ⁇ l.
  • VSVG vesicular stomatitis virus glycoprotein
  • I.U. infectious units
  • mice Female mice (C57Bh/6, 6D2F1 ; IFFA, Credo, France or NMRI Charles River France) aged 5 to 8 weeks were stimulated (day 1) with one peritoneal injection of follicle stimulating hormone (FSH, 5-10u, Folligon; Intervet AG. , Pfaffikon, Switzerland) , followed on day 3 by a second injection (10 IU/0.2 ml) of human chorionic gonadotrophin (HCG, Pregnyl ; Organon, Zurich, Switzerland) to induce ovulation. Females were then mated with normal males from the same strain. The day after the females were killed by cervical dislocation 13 h after HCG administration.
  • FSH follicle stimulating hormone
  • HCG human chorionic gonadotrophin
  • the swollen ampullae of the oviducts were dissected; the available oocyte-cumulus complexes were isolated under a stereo microscope in M2 medium containing hyaluronidase (300 ⁇ g/ml) and maintained under standard incubation conditions (10% C0 2 ) .
  • the thus prepared oocytes can then be submitted to the zona pellucida laser-drilling procedure.
  • said procedure is described in detail:
  • each oocyte was positioned to bring a region of the zona pellucida on the aiming spot and the zona pellucida was exposed once or twice to 1-2 ms laser light .
  • the diameter of the drilled holes varied between 2-20 ⁇ m.
  • the appropriately prepared and drilled oocytes are brought into contact with the lentiviral vectors. This can be done by incubating the drilled fertilised oocytes with 3 to 5 , preferably 4 hours with lentiviruses carrying a particular transgene .
  • the transgene can be any transgene of interest, specifically those which will introduce a desired property into a host.
  • the embryo After incubation which is preferably carried out at 20 to 37°C, more preferred at 37°C, under standard incubation conditions (10% C0 2 ) the embryo can be transferred back to foster mothers the same day.

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Abstract

La présente invention concerne l'utilisation d'un laser 1,48 µm pour réaliser la microperforation d'un orifice dans la zona pellucida d'un embryon monocellulaire. Ainsi, un rétrovirus, en particulier un lentivirus portant un transgène particulier, peut être introduit dans ledit embryon.
EP04708744A 2003-02-06 2004-02-06 Microperforation sans contact de zona pellucida d'ovocytes avec un faisceau laser a diode de 1,48 m pour l'introduction de retrovirus Withdrawn EP1589811A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US44588503P 2003-02-06 2003-02-06
US445885P 2003-02-06
PCT/EP2004/001101 WO2004069993A2 (fr) 2003-02-06 2004-02-06 MICROPERFORATION SANS CONTACT DE ZONA PELLUCIDA D'OVOCYTES AVEC UN FAISCEAU LASER A DIODE DE 1,48 µM POUR L'INTRODUCTION DE RETROVIRUS

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EP1589811A2 true EP1589811A2 (fr) 2005-11-02

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EP04708744A Withdrawn EP1589811A2 (fr) 2003-02-06 2004-02-06 Microperforation sans contact de zona pellucida d'ovocytes avec un faisceau laser a diode de 1,48 m pour l'introduction de retrovirus

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WO (1) WO2004069993A2 (fr)

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CA3021323A1 (fr) 2016-04-20 2017-10-26 Coopersurgical, Inc. Orientation de faisceau pour systemes laser et procedes associes

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WO2004069993A2 (fr) 2004-08-19

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