Electroporation-mediated genome editing in vitrified/warmed mouse zygotes created by IVF via ultra-superovulation

Experimental Animals
Yoshiko NakagawaTakashi Yamamoto

Abstract

Recently, genome editing in mouse zygotes has become convenient and scalable, in association with various technological developments and improvements such as novel nuclease tools, alternative delivery methods, and contemporary reproductive engineering techniques. We have so far demonstrated the applicability of ultra-superovulation, in vitro fertilization (IVF), and vitrification/warming of zygotes in microinjection-mediated mouse genome editing. Moreover, an electroporation-mediated method has rapidly become established for simple gene knockout and small precise modifications including single amino acid substitutions. Here, we present an updated example of an application coupling the following three latest technologies: 1) CRISPR-Cas9 ribonucleoprotein as the most convenient genome-editing reagent, 2) electroporation as the most effortless delivery method, and 3) cryopreserved oocytes created by IVF via ultra-superovulation as the most animal welfare- and user-friendly strategy. We successfully created gene knockout and knock-in mice carrying insertion/deletion mutations and single amino acid substitutions, respectively, using the streamlined production system of mouse genome editing described above, referred to as the CREATRE...Continue Reading

References

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Citations

Mar 12, 2020·Current Protocols in Mouse Biology·Almudena FernándezLluís Montoliu
Sep 27, 2020·In Vitro Cellular & Developmental Biology. Animal·Maki HirataTakeshige Otoi
Sep 24, 2020·Laboratory animal research·Toru TakeoNaomi Nakagata
May 1, 2021·Frontiers in Genetics·Jason C Lin, Alison L Van Eenennaam
May 8, 2021·Frontiers in Genetics·Vanessa ChenouardIgnacio Anegon

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Methods Mentioned

BETA
PCR
electrophoresis
cesarean section
gene knockout

Software Mentioned

CREATRE
COSMID
ssODN

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