Nov 18, 2025

Public workspaceGeneration of transgenic rats using piggyBac transposase mRNA and piezo-assisted microinjection

PLOS One
DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpmnwdgzp/v1
  • Kohtaro Morita1,2
  • 1Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University;
  • 2RIKEN BioResource Research Center
  • Kohtaro Morita
kohtaro.morita
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpmnwdgzp/v1
External link: https://doi.org/10.1371/journal.pone.0339406
Protocol Citation: Kohtaro Morita 2025. Generation of transgenic rats using piggyBac transposase mRNA and piezo-assisted microinjection. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpmnwdgzp/v1
Manuscript citation:
Morita K, Ihashi S, Okamura E, Goto K, Yoshihara T, Honda A, Ema M, Asano M (2026) Highly efficient production of transgenic rats with long DNA insertions using piggyBac transposase mRNA and piezo-assisted microinjection. PLOS One 21(2). doi: 10.1371/journal.pone.0339406
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 17, 2025
Last Modified: November 18, 2025
Protocol Integer ID: 232785
Keywords: using piggybac transposase mrna, generating transgenic rat, piggybac transposase mrna, efficient production of transgenic animal, optimized piggybac transposase, transgenic rat, transgenic animal, piggybac transposase, embryo transfer, embryo transfer into pseudopregnant female, preparation of mammalian codon, mammalian codon, piggybac system, assisted pronuclear microinjection, pronuclear microinjection, assisted microinjection, mrna
Funders Acknowledgements:
JSPS KAKENHI
Grant ID: JP21H02388
JSPS KAKENHI
Grant ID: JP18H04883
Kyoto Univrsity Foundation
Abstract
We present a protocol for generating transgenic rats using the piggyBac system. It covers preparation of mammalian codon-optimized piggyBac transposase (mPBase) mRNA and donor plasmid DNA, superovulation and zygote collection, piezo-assisted pronuclear microinjection, and embryo transfer into pseudopregnant females, enabling efficient production of transgenic animals.
Materials
Animals
  • Adult females (8–15 weeks old)
  • Adult males (over 10 weeks old)

Plasmids and mRNA
  • Donor plasmid: transgene between ITRs
  • mPBase expression plasmid: pcDNA3.1-EGFP-poly(A)83 containing mPBase cDNA

Reagents

  • Xho I
  • MEGAscript T7 Transcription Kit (Thermo Fisher Scientific; AM1333)
  • Cap Analog (m7G(5’)ppp(5’)G) (Thermo Fisher Scientific; AM8048)
  • RNeasy Mini Kit (QIAGEN; 74104)
  • Saline
  • [des-Gly10, D-Ala6]-LH-RH ethylamide acetate salt hydrate (Sigma-Aldrich)
  • Pregnant mare serum gonadotropin (PMSG; Aska Animal Health)
  • Anti-inhibin serum (Central Research)
  • Human chorionic gonadotropin (hCG; Aska Pharmaceutical)
  • HTF medium (ARC Resource)
  • EmbryoMax Advanced KSOM Embryo medium (Advanced KSOM) (Merck)
  • Rat KSOM (ARK Resource)
  • Three types of mixed anesthetic agents ((0.375 mg/kg medetomidine, 2.0 mg/kg midazolam, and 2.5 mg/kg butorphanol)
  • Antisedan (Zenoaq)

Equipment

  • Inverted microscope (IX73, Olympus)
  • PMM4 Piezo impact drive unit (Prime Tech)
  • Micromanipulator (Narishige)
  • Incubator
  • Hot plate
Troubleshooting
Safety warnings
This protocol involves the use of animals and requires prior approval from the user's Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Ethics statement
The protocol was approved by the Animal Experimentation Committee and Recombinant DNA Experiment Safety Committee of Kyoto University (Med Kyo 22625, 23016, and 240401).
Preparation of donor plasmid DNA and mPBase mRNA
Prepare donor plasmid DNA at 10 ng/µL in nuclease-free water.
Linearize pcDNA3.1-EGFP-poly(A)83 containing mPBase cDNA using Xho I by incubating at 37°C overnight.
Incubation
Digestion
Overnight
Purify the linearized plasmid DNA by phenol-chloroform-ethanol precipitation.
Centrifigation
Synthesize mRNA using MEGAscript T7 Transcription Kit and Cap Analog according to manufacturer’s instructions.
Purify the synthesied mRNA using according to manufacturer’s instructions.
Centrifigation
Prepare mPBase mRNA at 100 ng/µL in nuclease-free water.
Mix equal volumes of donor plasmid DNA (final concentration 5 ng/µL) and mPBase mRNA (final concentration 50 ng/µL).
Mix
Superovulation and collection of rat zygotes
Superovulation is performed following the method described in [1], with slight modifications.
Inject adult females intraperitoneally with 0.04 mg [des-Gly10, D-Ala6]-LH-RH in 200 µL saline (day 0, approximately 11:00 AM).
48–50 h later (day 2, 11:00 AM–1:00 PM), inject PMSG (150 IU/kg) and anti-inhibin serum (100 µL).
48 h after PMSG/anti-inhibin (day 4, 11:00 AM to 1:00 PM), inject hCG (75 IU/kg).
Mate hCG-injected females with males the same day.
The next day, collect pronuclear-stage embryos from mated females 20–26 h after mating (day 5, 9:00 AM – 1:00 PM).
Culture zygotes in HTF medium at 37°C under 5% CO₂ prior to microinjection.
Incubation
Microinjection of mPBase mRNA and donor plasmid into pronuclei of zygotes
Prepare drops of Advanced KSOM, the mPBase mRNA/donor plasmid DNA mixture, and 12% PVP in Advanced KSOM on a 100-mm dish lid and cover the drops with paraffin oil.



Using an inverted microscope equipped with a PMM4 Piezo impact drive unit and a micromanipulator, microinject the mixture into the male pronuclei of zygotes in Advanced KSOM. Injections can be started approximately between 3:00 PM and 6:00 PM.







After injection, place zygotes at room temperature for 5 min.
Culture the injected zygotes in Rat KSOM (approximately 50 embryos/50 µL) at 37°C under 5% CO₂ until 2-cell stage.
Incubation
Embryo transfer
Embryo transfer is performed following the method described in [2].
Transfer 10–15 embryos per oviduct into pseudopregnant recipient females anesthetized with a mixture of three agents (0.5 mL/100 g body weight).
After transfer, administer 3% Antisedan at same volume as anesthetic.
Keep recipient female on 37°C hot plate for 3 h.
After 22 days of the embryo transfer, pups are delivered naturally or by caesarean section and fostered to surrogate mothers for care.
Protocol references
1. Honda A, Tachibana R, Hamada K, Morita K, Mizuno N, Morita K, Asano M. Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization. Sci Rep. 2019 Aug 9;9(1):11571. doi: 10.1038/s41598-019-47964-1. Erratum in: Sci Rep. 2020 Jan 30;10(1):1830. doi: 10.1038/s41598-020-58515-4.

2. Morita K, Honda A, Asano M. A Simple and Efficient Method for Generating KO Rats Using In Vitro Fertilized Oocytes. Methods Mol Biol. 2023;2637:233-246. doi: 10.1007/978-1-0716-3016-7_18.