Lasergestützte Zytoplasmatischen Mikroinjektions in Vieh Zygotes
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
Zytoplasmatischen Mikroinjektion von 1-Zelle Embryonen ist eine sehr leistungsfähige Technik. Es kann für die Bereitstellung jede Lösung in den Embryo, beispielsweise verwendet werden, Gen-Knock-outs erzeugen Genfunktion zu untersuchen oder Gen-edited Tiere zu erzeugen. Die meisten landwirtschaftlich relevanten Nutztier Zygoten haben eine sehr hohe Fettsäurezusammensetzung, die ihr Zytoplasma opak und dunkel 1 macht. Sie haben auch eine ziemlich elastische Plasmamembran (PM). Diese Eigenschaften machen die Mikroinjektion unter Verwendung von herkömmlichen pronukleären / Zytoplasma-Injektion wie bei Nagetieren herausfordernd und oft ungenau verwendet.
Zytoplasmatischen Mikroinjektions hat Vorteile gegenüber pronukleären Mikroinjektions da es einfacher ist , durchzuführen und verursacht auch weniger Schaden an den injizierten Embryos, in höheren Lebensfähigkeit resultierende 2. Das übergeordnete Ziel dieses Protokolls ist es, eine erfolgreiche Methode für die Bereitstellung von Lösungen in das Zytoplasma von landwirtschaftlichen Nutztieren Zygoten zu demonstrieren. Zu können auszuführenzytoplasmatischen Mikroinjektions mit hoher Effizienz auf Nutztieren Embryonen wird ein Laser zur Erzeugung eines Lochs in der Zona pellucida (ZP) und dann eine blunt-end Glasnadel für die Mikroinjektion verwendet. Diese Strategie zielt darauf ab, die mechanische Schäden am Embryo während der Injektion eingeprägt zu reduzieren. Dann Aspiration von zytoplasmatischen Inhalten in der Injektionsnadel erlaubt, effizient und sicher ein Bruch des PM zu gewährleisten, dass die Lösung in das Cytoplasma des Embryos geliefert wird.
Diese Technik wurde in Rinderembryonen zu liefern siRNA in die zygotic Zytoplasma 3,4 und erzeugen Mutationen mit Hilfe der Cluster regelmäßig voneinander beabstandete kurze Palindrom Wiederholungen (CRISPR) / CRISPR zugehörige System 9 (Cas9) System 5 bereits erfolgreich eingesetzt. Es ist auch geeignet (mit geringfügigen Modifikationen) bovine Cumulus-umschlossenen Oozyten 6 einzuspritzen. Hier haben wir unsere Injektionsprotokoll liefert einen Farbstoff beschreiben kann, dass anwendbar sein, jede des zu injizierendenired Lösung in die Zygote, und zeigen, dass diese Technik unter Verwendung minimaler Lyse verursacht und beeinflusst nicht die frühe Embryoentwicklung.
Protocol
Representative Results
Discussion
Mikroinjektion von Zygoten ist ein gut eingeführtes Verfahren für Lösungen in Säugetierembryonen einzuführen. Mit einigen Variationen in Abhängigkeit von der Art und dem Ziel des Experiments kann diese Technik allgemein verwendet werden. Wir zeigen, wie intrazytoplasmatischer Mikroinjektions auszuführen unter Verwendung eines Lasers den Eingang eines stumpfen Enden Mikropipette zu unterstützen. Zygotes bestimmter Tierarten (wie Rinder, Schafe und Schwein) haben einen dunklen Zytoplasma, die Visualisierung der In…
Declarações
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. |
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