Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

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

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....

Protocol

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 it on a microforge in a horizontal position.
   4. Bring the pulled pipette and the microforge heater filament with its glass bead into focus. Us....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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.
100 x 15 mm culture dish	Falcon	351029	Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micromanipulator easier. The lid of a 60 mm culture dish can also be used.
35 mm culture dish	Corning	430165	These dishes are used for culturing the embryos in 50 μl 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 hr 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 10 mg/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 4 mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hr 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: 107.7 mM. Component of SOF-HEPES medium.
KCl	Sigma	P5405	Final concentration: 7.16 mM. Component of SOF-HEPES medium.
KH2PO4	Sigma	P5655	Final concentration: 1.19 mM. Component of SOF-HEPES medium.
MgCl2 6H2O	Sigma	M2393	Final concentration: 0.49 mM. Component of SOF-HEPES medium.
Sodium DL-lactate	Sigma	L4263	Final concentration: 5.3 mM. Component of SOF-HEPES medium.
CaCl2-2H2O	Sigma	C7902	Final concentration: 1.71 mM. Component of SOF-HEPES medium.
D-(−)-Fructose	Sigma	F3510	Final concentration: 0.5 mM. Component of SOF-HEPES medium.
HEPES	Sigma	H4034	Final concentration: 21 mM. 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: 4 mM. Component of SOF-HEPES medium.
Sodium pyruvate	Sigma	P4562	Final concentration: 0.33 mM. Component of SOF-HEPES medium.
Glutamax	Gibco	35050	Final concentration: 1 mM. Component of SOF-HEPES medium.
BSA	Sigma	A-3311	Final concentration: 1 mg/ml. Component of SOF-HEPES medium.
Gentamicin	Sigma	G-1397	Final concentration: 5 μg/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 22 μm filter can be stored in the fridge at 4 °C for 1 month. Warm in 37 °C water bath before use.