家畜接合体におけるレーザーアシスト細胞質マイクロインジェクション
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
1細胞胚の細胞質マイクロインジェクションは非常に強力な手法です。これは、例えば、遺伝子機能を研究するために、または遺伝子編集動物を生成するために遺伝子ノックアウトを生成するために胚に任意の溶液を送達するために使用することができます。ほとんどの農学関連の農場の動物の接合体は、それらの細胞質が不透明と1ダークになり、非常に高い脂肪酸組成を有します。彼らはまた、かなり弾性原形質膜（PM）を有します。これらの特性は、挑戦的で、しばしば不正確なげっ歯類で使用されるような従来の前核/細胞質の注入を使用して、マイクロインジェクションを行います。
実行が容易であり、また、より高い生存率を2で、その結果、注入された胚にはあまりダメージを引き起こすため、細胞質マイクロインジェクションは、前核マイクロインジェクションを超える利点を有しています。このプロトコルの全体的な目標は、農場の動物の接合体の細胞質にソリューションを提供するための成功した方法を実証することです。実行できるようにするには家畜の胚に高い効率で細胞質マイクロインジェクション、レーザは透明帯（ZP）に穴を生成するために使用され、その後、平滑末端ガラス針をマイクロインジェクションのために使用されます。この戦略は、注入中の胚に刻印機械的損傷を軽減することを目指しています。その後、注射針の内側の細胞質内容物の吸引は、溶液は、胚の細胞質に送達されることを確実にPMを効率的かつ自信を持って破損することができます。
この技術はすでに正常接合体の細胞質3,4へのsiRNAを送達するようにし、クラスタ化された定期的interspaced短いパリンドローム反復（CRISPR）/ CRISPR関連するシステム9（Cas9）システム5を用いて、突然変異を生成するために、ウシの胚で使用されてきました。また、ウシ卵丘に囲まれた卵母細胞6を注入する（若干修正済み）が適しています。ここでは、色素を提供我々の注入プロトコルを説明し、それは任意のDESの注入に適用することができますIRED接合子への溶液、およびこの技術を使用して最小限の溶解を引き起こし、初期胚の発育に影響を及ぼさないことを示しています。
Protocol
Representative Results
Discussion
接合体のマイクロインジェクションは、哺乳動物の胚にソリューションを導入するための十分に確立された方法です。種および実験の目的に応じていくつかのバリエーションでは、この技術は、広く使用することができます。私たちは、平滑末端マイクロピペットの入り口を支援するためにレーザーを使用して細胞質内マイクロインジェクションを実行する方法を示しています。 （例えばウ…
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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