Microinjection of mRNA into
fertilized or cloned zygotes
Bioresource Research Center
Bioresource Engineering Division
protocol #1

2020/05/20

1Graphical Abstract

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Contents• Introduction• Reagents and Equipment• Methods
1.
2.
3.
4.
5.
6.

Preparation of culture medium and mRNA
Preparation of microinjection needles
Preparation of a dish for microinjection
Preparation of fertilized or cloned zygotes
Microinjection of mRNA into zygotes
Culture of injected zygotes• References• Appendix• Contact3

Introduction
PurposeOverexpress a gene of interest by microinjection ofmRNA into fertilized or cloned zygotes.

Introduction
The mRNA of interest is injected into a fertilized orcloned embryo at one cell stage using amicromanipulator.

4Reagents and Equipment
Glass pipette

Sutter Instrument
Drummond

B100-75-10
1-000-0500

PullerSutter Instrument

P-97/IVF

Microforge

NARISHIGE

MF-900Micromanipulator with PIEZO

Nikon, Prime tech, etc.

Dishes for micromanipulation

Depends on the microscope
systemDishes for embryo culture

IWAKI

Embryo culture medium (KSOM, CZB,Homemadeetc.)

1010-060
1000-035
HomemadeMedium for micromanipulation(Hepes-KSOM, Hepes-CZB, M2medium, etc.)

Homemade or
MERCK

Homemade or
MR-015-D

10%PVP

Homemade or
Irvine Scientific

Homemade or90123 5

Methods
1. Preparation of culture medium and mRNA
• Culture medium
For long-term culture: KSOM or CZB, etc.
For micromanipulation: Hepes-KSOM, Hepes-CZB, M2 medium, etc.
For pipette washing: 10% PVP• mRNAFor mRNA synthesis, see protocol #X. mRNA is dissolved in nuclease freewater and aliquots were stored at -80℃. Thaw just before use.
For the purification of mRNA for microinjection, ethanol-precipitation with
LiCl is usually OK. However, if the embryo is highly sensitive to the purity ofmRNA (e.g., marmoset), it is better to purify it with a column such as theMEGA clear kit.
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Methods
2. Preparation of microinjection needles
Pull a glass pipette with a puller and cut the tip so that the outer
diameter of the tip is 4-5um. Then, heat the tip with a forge (close tothe glass ball) to make it as round and smooth as possible. Inject
mercury or its substitute (such as Fluorinert) into the pipette (1-2mm in length).
Mercury

Glass pipette filled with mercury
Rounded glass pipette tip7

Methods
3. Preparation of a dish for microinjection
The dish should be prepared according to the microscope systemused. (We make homemade glass bottom dishes.)Three types of drops: 10% PVP for pipette washing, culture medium
for embryo manipulation, and mRNA are arranged as shown in thefigure. Finally, cover the drops with mineral oil.
10%PVP
Culture mediummRNA
Homemade glass bottom dish8

Methods
4. Preparation of fertilized or cloned zygotesFertilized embryos or cloned embryos are prepared as pronuclear
stage embryos 5-6 hours after fertilization or activation, respectively.
This timing is arbitrary and unfertilized eggs and even later embryos(e.g., at the two-cell stage) can be injected, too. However, thesurvival rate after injection is higher in post-activated embryos thanunfertilized eggs because they are more resistant to piezoelectric
damage.

Methods
5. Microinjection of mRNA into zygotes
First, the embryos are transferred to the culture medium for embryo
manipulation. Wash glass pipettes attached to micromanipulatorswith 10% PVP and mercury. Fill the pipette to the tip with mercury,move to the mRNA drop and suck a suitable amount of mRNA into
the pipette (usually the equivalent of 5-10 injections). Return to theculture medium for embryo manipulation and hold the embryos witha holding pipette. Make a hole in the zona pellucida with a strong
piezo. Then, the cytoplasmic membrane is pushed in with a pipette
while avoiding the pronuclei. When the tip of the pipette reaches
near the other side of membrane, break the membrane with a weakpiezo. mRNA is injected into the cytoplasm in the right amount(about 10 pl for an amount of one anterior nucleus). After 5-10
injections, suck the mRNA again and repeat the injections.

Methods
6. Culture of injected zygotes
After injection, the embryos are allowed to rest in the culture
medium for embryo manipulation for 10-15 min (returning to theincubator immediately after injection will reduce the survival rate).
The embryos are then transferred to the normal culture medium forlong-term incubation.

References• Matoba S, Nakamuta S, Miura K, Hirose M, Shiura H, Kohda T, Nakamuta N, Ogura
A. Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated withintrauterine growth restriction in mice. Proc Natl Acad Sci U S A. 2019 Oct
15;116(42):21047-21053.
• Miura K, Matoba S, Ogonuki N, Namiki T, Ito J, Kashiwazaki N, Ogura A. Application ofauxin-inducible degron technology to mouse oocyte activation with PLCζ. J Reprod
Dev. 2018 Aug 20;64(4):319-326.
• Matoba S, Liu Y, Lu F, Iwabuchi KA, Shen L, Inoue A, Zhang Y. Embryonic development
following somatic cell nuclear transfer impeded by persisting histone
methylation. Cell. 2014 Nov 6;159(4):884-95.
• Inoue K, Matoba S, Ogura A. Somatic Cell Nuclear Transfer in Mice: Basic Protocol andIts Modification for Correcting X Chromosome Inactivation Status. Methods Mol
Biol. 2018;1861:55-65.
12Appendix

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Contact• Bioresource Engineering Division• Question

Shogo Matoba: shogo.matoba@riken.jp

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