Asistida por láser citoplasmática microinyección en el ganado cigotos
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
microinyección citoplasmática de embriones de 1 célula es una técnica muy poderosa. Puede ser utilizado para la entrega de cualquier solución en el embrión, por ejemplo, producir genes knock-outs para estudiar la función de genes o de genes para generar animales editado. Cigotos de animales de granja agrícolas más relevantes tienen una composición de ácidos grasos muy alto que hace que su citoplasma opacos y oscuros 1. También tienen una membrana plasmática bastante elásticas (PM). Estas características hacen que la microinyección pronuclear mediante inyección convencional / citoplasmática como se utiliza en especies de roedores difíciles y, a menudo inexactos.
Microinyección citoplasmática tiene ventajas sobre la microinyección pronuclear, ya que es más fácil de realizar y también causa menos daño a los embriones inyectados, resultando en una mayor viabilidad 2. El objetivo general de este protocolo es demostrar un método exitoso para la entrega de soluciones en el citoplasma de cigotos de animales de granja. Para ser capaz de realizarmicroinyección citoplásmica con una alta eficiencia en embriones de ganado, se utiliza un láser para generar un agujero en la zona pelúcida (ZP) y luego una aguja de vidrio de extremo romo se utiliza para la microinyección. Esta estrategia tiene como objetivo reducir el daño mecánico impreso en el embrión durante la inyección. Entonces, la aspiración del contenido citoplasmático dentro de la aguja de inyección permite la rotura eficiente y seguro de la PM asegurar que la solución se suministra en el citoplasma del embrión.
Esta técnica ya se ha utilizado con éxito en los embriones de bovino para entregar siRNA en el citoplasma cigóticos 3,4 y para generar mutaciones utilizando las repeticiones agrupadas espaciadas regularmente cortos palindrómicas (CRISPR) / sistema CRISPR asociado 9 sistema 5 (Cas9). También es adecuado (con modificaciones menores) para inyectar la especie bovina ovocitos cumulus-cerrado 6. A continuación, describimos nuestro protocolo de inyección entrega de un medio de contraste, que puede ser aplicable a inyectar cualquier dessolución IRED en el zigoto, y muestran que el uso de esta técnica provoca la lisis mínima y no afecta el desarrollo embrionario temprano.
Protocol
Representative Results
Discussion
La microinyección de cigotos es un método bien establecido para la introducción de soluciones en embriones de mamíferos. Con algunas variaciones dependientes de la especie y el objetivo del experimento, esta técnica se puede utilizar ampliamente. Mostramos cómo realizar la microinyección intracitoplasmática de usar un láser para ayudar a la entrada de una micropipeta de extremos romos. Los cigotos de algunas especies de ganado (tales como ganado vacuno, ovejas y cerdos) tienen un citoplasma oscuro, dificultando…
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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