Generation of transgenic mouse lines
We have improved the protocol to efficiently establish relevant transgenic mouse lines by directly microinjecting any purified expression vectors or BACs into pronuclei of mouse fertilized eggs (Figure) and transferring them into pseudo-pregnant recipient mice (Inoue T et al., 2008). Components for genome editing (proteins, RNAs, and single/double strand DNAs; see below) may easily be introduced into fertilized eggs by means of electroporation. We have set up our own microinjection system usable any time in the well-organized specific pathogen-free animal facility of our institute.
Mammalian whole embryo culture system
Since early cellular and/or molecular events during mammalian development proceed in the deep inside of the uterus, it had been very difficult to directly visualize, trace and manipulate them. The mammalian whole embryo culture apparatus with rotating culture bottles and successive gas supply (Figure; Ikemoto Rika, Japan) has allowed us to completely recapitulate embryonic development at the post-implantation stages in vitro,if the embryos with extra-embryonic tissues are carefully and properly dissected out from the uterus (Figure). For example, mouse whole embryos can grow normally in this system for any 2~3 days from E7.0 to E13.0, duration of which covers most important events for early neural development such as neural induction, neurulation, brain regionalization (neuromere formation) and initial neuronal wiring processes. We have also established good methods for cell labeling (Inoue T et al., 2000; Inoue T et al., 2001) and electroporation-mediated gene delivery (Inoue T et al., 2001; Osumi N and Inoue T, 2001; Inoue T and Krumlauf R, 2001) in the system.
Genome editing by means of CRISPR/Cas9 system
We have quickly adopted and upgraded the recently emerging CRISPR/Cas9 mediated genome editing method for fertilized mouse eggs in the lab. We have so far successfully and resourcefully generated various kinds of gene knock-out (KO) mice (e.g. from one base frame shift mutation to ~90-kb whole locus deletion) as well as knock-in (KI) mouse lines (e.g. from one base non-synonymous replacement in the pathological SNP to ~2-kb integration of functional gene expression cassettes). Methodologically, we deliver the cloning-free RNA/Cas9 protein complex for simple KO mouse generation to minimize the mosaicism and for KI mice, we introduce the homologous region/arms containing single or double strand DNA with the RNA/Cas9 complex, depending on the situation. As for the delivery into fertilized mouse eggs, we carry out either pronuclear injection or the Genome editor (BEX, Japan) mediated square-pulsed-electroporation.