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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Genetics

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Published: October 5th, 2016

DOI:

10.3791/54465

1Department of Animal Science, University of California, Davis

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.

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.

Cytoplasmic microinjection of 1-cell embryos is a very powerful technique. It can be used for delivering any solution into the embryo to, for example, produce gene knock-outs to study gene function or to generate gene-edited animals. Most agriculturally-relevant farm animal zygotes have a very high fatty acid composition that makes their cytoplasm opaque and dark1. They also have a fairly elastic plasma membrane (PM). These characteristics make microinjection using conventional pronuclear/cytoplasmic injection as used in rodent species challenging and often inaccurate.

Cytoplasmic microinjection has advantages over pronuclear mic....

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1. Micropipette Production

  1. Injection micropipette
    1. Place a borosilicate glass capillary (outer diameter (OD): 1.0 mm, inner diameter (ID): 0.75 mm) in a micropipette puller (in the center of the right and left capillary holders) and lock it.
    2. Use an appropriate program to pull the glass capillary so it results in a thin tip with a long tapper. (Example: Heat: 825; Pull: 30; Velocity: 120; Time: 200; Pressure: 500).
    3. Carefully remove the pulled pipettes from the device and place .......

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Laser-assisted cytoplasmic microinjection is a powerful and reliable protocol to deliver solutions into the cytoplasm of livestock zygotes. Figure 3 shows a general outline of the zygotes before and after injection as well as the overall outline of the technique. Dextran-red is used as injecting solution to allow tracking site of injection and injection efficiency and accuracy. Successful delivery of the solution is illustrated in Figure 4 showing a recen.......

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Microinjection of zygotes is a well-established method for introducing solutions into mammalian embryos. With some variations dependent on the species and the aim of the experiment, this technique can be broadly used. We show how to perform intracytoplasmic microinjection using a laser to assist the entrance of a blunt-end micropipette. Zygotes of some livestock species (such as cattle, sheep, and pig) have a dark cytoplasm, hindering the visualization of the injection pipette once inside the embryo. Also, their plasma m.......

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Work related to this technique is supported by NIH/NICHD RO1 HD070044 and USDA/NIFA Hatch projects W-3171 and W-2112.

....

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Name Company Catalog Number Comments
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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  5. 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).
  6. 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).
  7. 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).
  8. Sutter Instrument. . Pipette Cookbook 2015 P-97 & P-1000 Micropipette Pullers. , (2015).
  9. Sutter Instrument. . P-97 Flaming/BrownTM Micropipette Puller Operation Manual Rev. 2.30 - DOM (20140825). , (2014).
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  11. 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).

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