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

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 (CARD-based Reproductive Engineering-Assisted Technology for RNP Electroporation) system. Owing to its accessibility, robustness, and high efficiency, we believe that our CREATRE protocol will become widely used globally for the production of genome-edited mice.

Single-Cell Transcriptome Profiling Revealed That Vitrification of Somatic Cloned Porcine Blastocysts Causes Substantial Perturbations in Gene Expression

“…Vitrification has become more and more popular in conservation of mammalian embryos because of its simplicity, speed, low cost, and high success rate. To date, vitrification of embryos in cattle (Taylor-Robinson et al, 2014 ), mouse (Nakagawa et al, 2018 ), and human (Wei et al, 2019 ) is becoming a routine procedure in facilitating the wide application of assisted reproductive technologies in these species. However, vitrification of porcine embryos still remains challenging.…”

Efficient production of immunodeficient non-obese diabetic/Shi-scid IL2rγnull mice via the superovulation technique using inhibin antiserum and gonadotropin

“…Additionally, IASe-derived fertilized oocytes at the pronuclear stage are used in gene modification through microinjection or electroporation. 31–33 In this study, we demonstrated that our optimized superovulation protocol for NOG mice was effective in efficiently and promptly expanding a colony of genetically engineered NOG mice. Additionally, an efficient production and storage system of NOG mice was developed through embryo and sperm cryopreservation.…”

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models

“…In some studies, electroporation was done in mouse zygotes that were denuded of the zona pellucida (ZP) by a Tyrod's acid treatment (Qin et al, 2015;Chen et al, 2016;Wang et al, 2016), without affecting the early development unlike data reported in rats (Okuyama and Funahashi, 2012). Electroporation also can be applied to mouse and rat frozen zygotes for efficient introduction of CRISPR RNP complexes, without affecting embryo viability or development (Nakagawa et al, 2018;Kaneko and Nakagawa, 2020).…”