Generation of Pax6-IRES-EGFP knock-in mouse via the cloning-free CRISPR/Cas9 system to reliably visualize neurodevelopmental dynamics. (July 2018)

• Record Type: Journal Article
• Authors: Inoue, Yukiko U.; Morimoto, Yuki; Hoshino, Mikio; Inoue, Takayoshi
• Is Part Of: Neuroscience research. Volume 132(2018)
• Page Start: 1
• Page End: 7
• Publication Date: 2018-07
• Subjects: Pax6 -- IRES -- EGFP -- CRISPR/Cas9 -- knock-in mouse; Neurosciences -- Research -- Periodicals; Neurosciences -- Research -- Japan -- Periodicals; Neurology -- Periodicals; Neurosciences -- Periodicals; Neurosciences -- Recherche -- Périodiques; Neurosciences -- Recherche -- Japon -- Périodiques; Neurosciences -- Research; Japan; Periodicals; 612.8
• DOI: 10.1016/j.neures.2018.01.007
• Languages: English
• ISSNs: 0168-0102

Abstract

Highlights: We generate Pax6-IRES-EGFP knock-in mouse by the cloning-free CRISPR/Cas9 system. EGFP expressions in the knock-in mice recapitulate the endogenous Pax6 expressions. At embryonic stages, Pax6-expressing regions are detectable without PCR-genotyping. Visualization of Pax6 expression dynamics by this line offers useful research tools. Abstract: Pax6 encodes a transcription factor that plays pivotal roles in eye development, early brain patterning, neocortical arealization, and so forth. Visualization of Pax6 expression dynamics in these events could offer numerous advantages to neurodevelopmental studies. While CRISPR/Cas9 system has dramatically accelerated one-step generation of knock-out mouse, establishment of gene-cassette knock-in mouse via zygote injection has been considered insufficient due to its low efficiency. Recently, an improved CRISPR/Cas9 system for effective gene-cassette knock-in has been reported, where the native form of guide RNAs (crRNA and tracrRNA) assembled with recombinant Cas9 protein are directly delivered into mouse fertilized eggs. Here we apply this strategy to insert IRES-EGFP -pA cassette into Pax6 locus and achieve efficient targeted insertions of the 1.8 kb reporter gene. In Pax6-IRES-EGFP mouse we have generated, EGFP-positive cells reside in the eyes and cerebellum as endogenous Pax6 expressing cells at postnatal day 2. At the early embryonic stages when the embryos are transparent, EGFP-positive regions can be easily identified without PCR-based genotyping, precisely recapitulating the endogenous Pax6 expression patterns. Remarkably, at E12.5, the graded expression patterns of Pax6 in the developing neocortex now become recognizable in our knock-in mice, serving a sufficiently sensitive and useful tool to precisely visualize neurodevelopmental processes.