Lazer destekli Hayvancılık Zigotlann Sitoplazmik Mikroenjeksiyon
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-hücre embriyoların Sitoplazmik mikroenjeksiyon çok güçlü bir tekniktir. Bu, örneğin, gen fonksiyonu çalışma ya da gen düzenlenmiş hayvanlar oluşturmak için, gen, çıkıntılara üretilmesi için embriyo herhangi bir çözüm sunmak için de kullanılabilir. En tarımsal-ilgili çiftlik hayvanı zigot kendi sitoplazma opak ve 1 karanlık kılan çok yüksek yağ asidi kompozisyonu var. Onlar da oldukça esnek plazma zarı (PM) var. Bu özellikler kemirgen türleri zorlu ve sık sık yanlış kullanılan geleneksel pronüklear / sitoplazmik enjeksiyon kullanılarak mikroenjeksiyon yapmak.
Sonuçlar, daha kolay olan ve aynı zamanda daha yüksek canlılığı 2'de elde edilen, enjekte edilen embriyolar için daha az zarar yana Sitoplazmik mikroenjeksiyon pronükleer mikroenjeksiyon fazla avantajlara sahiptir. Bu protokolün genel amacı çiftlik hayvanı zigot sitoplazması içine çözümler sunmak için başarılı bir yöntem ortaya koymaktır. yapabilmek içinBesi embriyoların yüksek verimle sitoplazmik mikroenjeksiyon, bir lazer, bir küt uç cam iğne mikroenjeksiyon için kullanılan zona pellucida (ZP) ve daha sonra bir delik oluşturmak için kullanılır. Bu strateji enjeksiyon sırasında embriyo üzerinde baskılı mekanik zararı azaltmayı hedefliyor. Sonra, enjeksiyon iğnesi içinde sitoplazmik içeriğin aspirasyon çözümü embriyo sitoplazmaya teslim edilmesini sağlamak PM verimli ve güvenli kırılmasına izin verir.
Bu teknik başarıyla zigotik sitoplazma 3,4 içine siRNA teslim etmek ve kümelenmiş düzenli interspaced kısa palindromik tekrarı (CRISPR) / CRISPR ilişkili sistem 9 (Cas9) sistemi 5 ile mutasyon üretmek için sığır embriyolarının kullanılmıştır. Ayrıca sığır cumulus-kapalı oosit 6 enjekte etmek (küçük değişikliklerle) uygundur. Burada, bir boya teslim eden enjeksiyon protokol açıklar, herhangi des enjekte uygulanabilir olabilirzigot içine IRED çözüm ve bu tekniği kullanarak en az erimesine yol açar ve erken embriyo gelişimini etkilemediğini göstermektedir.
Protocol
Representative Results
Discussion
zigot Mikroenjeksiyon memeli embriyolarının içine çözümleri tanıtmak için köklü bir yöntemdir. türleri ve Deneyin amacı göre bazı farklılıklar ile, bu teknik genel olarak kullanılabilir. Biz bir künt uç mikropipet girişinde yardımcı olmak için bir lazer kullanarak intrasitoplazmik mikroenjeksiyon nasıl gerçekleştirileceğini gösterir. (Örneğin sığır, koyun ve domuz gibi) bazı hayvan türlerinin zigot, embriyo içinde bir kez enjeksiyon pipet görselleştirme engelleyen, karanlık bir si…
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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