Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout...

Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs into the zygotes ex vivo, and (3) surgically transferring them into the oviduct of pseudo-pregnant females. This process requires a significant number of animals, as it involves not only donor females but also vasectomized males and pseudo-pregnant females. Moreover, microinjections into the cytoplasm or pronucleus of mouse zygotes present challenges such as needle clogging, membrane permeability issues due to high elasticity, and potential embryo death. The development of advanced electroporators, such as the Nepa21, provides a unique opportunity to generate mice with targeted gene knockouts in a single step through a method known as Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD). This technique involves microinjecting CRISPR-Cas components (Cas9 protein and guide RNA) into the oviducts of pregnant females at 0.7 days post-conception, followed by in vivo electroporation to deliver these components directly into the zygotes. Following the I-GONAD procedure, the pregnant mouse carries and gives birth to pups with the targeted gene knockout. This article provides a detailed, step-by-step protocol for implementing the I-GONAD method in mice, offering a more efficient and accessible alternative to traditional knockout mouse generation techniques.