레이저를 이용한 가축 접합체의 세포질 미세 주입을
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 셀 배아의 세포질 미세 주입은 매우 강력한 기술이다. 또, 예를 들면, 유전자 기능을 연구 나 유전자 편집 동물을 생성하는 유전자가 녹아웃을 제조 배아에 어떤 솔루션을 제공에 사용될 수있다. 대부분의 농업 관련 농장 동물 접합자는 세포질이 불투명 한 어두운 만드는 매우 높은 지방산 조성. 또한 상당히 탄성 원형질막 (PM)을 갖는다. 이러한 특성은 설치류 종의 도전과 종종 부정확에 사용되는 기존의 전핵 / 세포질 주입을 사용하여 미세 주입을합니다.
이를 수행하기가 쉽습니다 또한 더 높은 생존 능력이 결과, 주입 된 배아에 덜 손상이 발생하기 때문에 세포질 마이크로 인젝션은 전핵 미세 주입을 통해 이점이있다. 이 프로토콜의 전반적인 목표는 농장 동물 접합체의 세포질에 솔루션을 제공하기위한 성공적인 방법을 설명하는 것입니다. 수행 할 수 있어야합니다가축 배아 고효율 세포질 마이크로 인젝션은 레이저는 평활 말단 유리 바늘이 미세 주입에 사용되는 투명대 (ZP) 다음에 구멍을 생성하는데 사용된다. 이 전략은 주입하는 동안 배아에 각인 기계적인 손상을 감소하는 것을 목표로하고있다. 그리고, 주사 바늘 내부 세포질 콘텐츠 흡인 용액 배아의 세포질 내로 전달되는 것을 보장 PM 효율적 자신감 파괴 할 수있다.
이 기술은 이미 성공적 접합체 세포질 3,4-으로 siRNA를 전달하고 클러스터 정기적 interspaced 짧은 상동 반복 (CRISPR) / CRISPR 연관된 시스템 (9) (Cas9) 시스템 (5)을 사용하여 돌연변이를 생성 소 태아에서 사용되어왔다. 또한 소 운 동봉 된 난 모세포 (6)를 주입하기 (약간의 수정과)에 적합하다. 여기서, 우리는 우리의 염료를 제공 주입 프로토콜을 설명하는 데 어떤 주입에 적용 할 수있다수정란 내로 IRED 용액 및이 기술을 사용하여 최소한의 용해가 발생 초기 배아 발달에 영향을주지 않는 것으로 나타났다.
Protocol
Representative Results
Discussion
접합체의 미세 주입은 포유 동물의 배아에 솔루션을 도입하기위한 잘 정립 된 방법이다. 종 및 실험 목적에 따라 일부 변형이 기술은 광범위하게 사용될 수있다. 우리는 평활 말단 마이크로 피펫의 입구를 돕기 위해 레이저를 사용하여 세포질 마이크로 인젝션을 수행하는 방법을 보여준다. (예 : 소, 양, 돼지 등) 일부 가축 종의 접합자는 배아의 내부에 한 번 주입 피펫의 시각화를 방해, 어두운 ?…
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
- McEvoy, T., Coull, G., Broadbent, P., Hutchinson, J., Speake, B. Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida. J Reprod Fertil. 118 (1), 163-170 (2000).
- Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A. 82 (13), 4438-4442 (1985).
- Ross, P. J., et al. Parthenogenetic activation of bovine oocytes using bovine and murine phospholipase C zeta. BMC Dev Biol. 8 (1), (2008).
- Canovas, J., Cibelli, J. B., Ross, P. J. Jumonji domain-containing protein 3 regulates histone 3 lysine 27 methylation during bovine preimplantation development. Proc Natl Acad Sci U S A. 109 (7), 2400-2405 (2012).
- Bogliotti, Y. S., et al. 4 Developmental outcomes and effiency of two CRISPR/Cas9 microinjection methods in bovine zygotes. Reprod Fertil Dev. 27 (1), 94-94 (2015).
- Bakhtari, A., Ross, P. J. DPPA3 prevents cytosine hydroxymethylation of the maternal pronucleus and is required for normal development in bovine embryos. Epigenetics. 9 (9), 1271-1279 (2014).
- Yaul, M., Bhatti, R., Lawrence, S. Evaluating the process of polishing borosilicate glass capillaries used for fabrication of in-vitro fertilization (iVF) micro-pipettes. Biomed Microdevices. 10 (1), 123-128 (2008).
- Sutter Instrument. . Pipette Cookbook 2015 P-97 & P-1000 Micropipette Pullers. , (2015).
- Sutter Instrument. . P-97 Flaming/BrownTM Micropipette Puller Operation Manual Rev. 2.30 – DOM (20140825). , (2014).
- Cibelli, J. B., Lanza, R. P., Campbell, K. H. S., West, M. D. . Principles of cloning. , (2002).
- Wang, B., et al. Expression of a reporter gene after microinjection of mammalian artificial chromosomes into pronuclei of bovine zygotes. Mol Reprod Dev. 60 (4), 433-438 (2001).
