Cytoplasmique microinjection dans zygotes Bétail assistée par laser
Summary
This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.
Abstract
Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.
Introduction
microinjection cytoplasmique d'embryons 1 cellules est une technique très puissante. Il peut être utilisé pour fournir une quelconque solution dans l'embryon, par exemple, produire des gènes knock-out pour étudier la fonction des gènes ou pour générer des animaux génétiquement modifiés. Zygotes des animaux d'élevage plus d'intérêt agronomique ont une composition en acides gras très élevé qui rend leur cytoplasme opaque et sombre 1. Ils ont aussi une membrane plasmique assez élastique (PM). Ces caractéristiques font de microinjection pronucléaire utilisant / injection cytoplasmique classique utilisé dans des espèces de rongeurs difficiles et souvent inexactes.
La micro – injection cytoplasmique présente des avantages sur la micro – injection pronucléaire car il est plus facile à réaliser et entraîne aussi moins de dommages aux embryons injectés, ce qui entraîne la viabilité élevée 2. L'objectif global de ce protocole est de démontrer une méthode efficace pour la fourniture de solutions dans le cytoplasme des zygotes des animaux d'élevage. Pour être en mesure d'effectuerla micro-injection cytoplasmique avec un rendement élevé sur des embryons de bétail, un laser est utilisé pour produire un trou dans la zone pellucide (ZP), puis une aiguille de verre à extrémité émoussée est utilisée pour la micro-injection. Cette stratégie vise à réduire les dommages mécaniques imprimé sur l'embryon lors de l'injection. Ensuite, l'aspiration du contenu cytoplasmique à l'intérieur de l'aiguille d'injection permet une rupture efficace et sûre de la MP en sorte que la solution est délivré dans le cytoplasme de l'embryon.
Cette technique a déjà été utilisée avec succès dans les embryons de bovins pour délivrer l' ARNsi dans le cytoplasme 3,4 zygotique et pour générer des mutations à l' aide des séquences répétées courtes regroupées régulièrement espacées (palindromiques CRISPR) / CRISPR associé du système 9 du système (cas9) 5. Il est également approprié (avec des modifications mineures) pour injecter bovins cumulus-ovocytes enfermés 6. Ici, nous décrivons notre protocole d'injection délivrant un colorant, qui peut être applicable à l'injection d'une quelconque dessolution IRED dans le zygote, et de montrer que l'utilisation de cette technique provoque la lyse minimale et n'a aucune incidence sur le développement précoce de l'embryon.
Protocol
Representative Results
Discussion
Microinjection de zygotes est une méthode bien établie pour l'introduction de solutions dans des embryons de mammifères. Avec quelques variations dépendant de l'espèce et du but de l'expérience, cette technique peut être utilisée au sens large. Nous montrons comment effectuer microinjection intracytoplasmique utilisant un laser pour aider l'entrée d'une micropipette extrémité franche. Zygotes de certaines espèces animales (comme les bovins, les moutons et les porcs) ont un cytoplasme somb…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Work related to this technique is supported by NIH/NICHD RO1 HD070044 and USDA/NIFA Hatch projects W-3171 and W-2112.
Materials
| Micropipette puller | Sutter Instrument | P-97 | |
| Glass capillary | Sutter instruments | B100-75-10 | These capillaries are used for making the holding and injecting pipettes. Any thick/standard wall borosilicate tubing without filament can be used. |
| Microforge | Narishige | MF-9 | Equipped with 10X magnification lense. |
| Micromanipulator | Nikon/ Narishige | NT88-V3 | |
| Inverted microscope | Nikon | TE2000-U | Equipped with 4x, 20x lenses and with a laser system. |
| Laser | Research Instruments | 7-47-500 | Saturn 5 Active laser. |
| Microdispenser | Drummond | 3-000-105 | The microdispenser is used to move the embryos. A p10 pipette can also be used but loading as minimal volume as possible. |
| 60mm culture dish | Corning | 430166 | Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micropipettes with the micromanipulator easier. |
| 35mm culture dish | Corning | 430165 | These dishes are used for culturing the embryos in 50μl drops covered with mineral oil. Alternatively, a 4 well dish can also be used. Regardless of the dish chosen to culture the embryos, they always have to be equilibrated in the incubator for at least 4 hours prior to transfering the embryos to them. |
| Incubator | Sanyo | MCO-19AIC | Any incubator that can be set to 38.5°C 5% CO2 conditions can be used. |
| Stereomicroscope | Nikon | SMZ800 | Used for visualizing the embryos in the culture drops and during washes. Any stereomicroscope with a 10x magnification can be used. |
| Control Unit HT | Minitube | 12055/0400 | Heating system attached to the stereomicroscope. |
| Heated Microscope Stage | Minitube | 12055/0003 | Heating system attached to the stereomicroscope. |
| Dextran-Red | Thermo Scientific | D1828 | A sterile 10mg/ml solution is used to inject. |
| Mineral Oil | sigma | M8410 | Keep the mineral oil at room temperature and protected from light using foil paper. |
| KSOMaa Evolve Bovine | Zenit | ZEBV-100 | Supplemented with 4mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hours before use. |
| FBS | Gemini-Bio | 100-525 | Use a stem-cell qualified FBS. |
| Zygotes | Zygotes are injected 17-20 hpf and can be in-vitro- or in-vivo-derived. | ||
| NaCl | Sigma | S5886 | Final concentration: 107.7mM. Component of SOF-HEPES medium. |
| KCl | Sigma | P5405 | Final concentration: 7.16mM. Component of SOF-HEPES medium. |
| KH2PO4 | Sigma | P5655 | Final concentration: 1.19mM. Component of SOF-HEPES medium. |
| MgCL2 6H2O | Sigma | M2393 | Final concentration: 0.49mM. Component of SOF-HEPES medium. |
| Sodium DL-lactate | Sigma | L4263 | Final concentration: 5.3mM. Component of SOF-HEPES medium. |
| CaCl2-2H2O | Sigma | C7902 | Final concentration: 1.71mM. Component of SOF-HEPES medium. |
| D-(−)-Fructose | Sigma | F3510 | Final concentration: 0.5mM. Component of SOF-HEPES medium. |
| HEPES | Sigma | H4034 | Final concentration: 21mM. Component of SOF-HEPES medium. |
| MEM-NEAA | Sigma | M7145 | Final concentration: 1X. Component of SOF-HEPES medium. |
| BME-EAA | Sigma | B6766 | Final concentration: 1X. Component of SOF-HEPES medium. |
| NaHCO3 | Sigma | S5761 | Final concentration: 4mM. Component of SOF-HEPES medium. |
| Sodium pyruvate | Sigma | P4562 | Final concentration: 0.33mM. Component of SOF-HEPES medium. |
| Glutamax | Gibco | 35050 | Final concentration: 1mM. Component of SOF-HEPES medium. |
| BSA | Sigma | A-3311 | Final concentration: 1mg/ml. Component of SOF-HEPES medium. |
| Gentamicin | Sigma | G-1397 | Final concentration: 5μg/ml. Component of SOF-HEPES medium. |
| Water for embryo transfer | Sigma | W1503 | Component of SOF-HEPES medium. |
| SOF-HEPES medium | Made in the lab | pH 7.3-7.4, 280±10 mOs. Filter sterilized through a 22μm filter can be stored in the fridge at 4° C for 1 month. Warm in 37 °C water bath before use. |
References
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