Figure 1
From: CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation
Construction and validation of CRISPR/Cas9 plasmids for Satb2 in HEK293T cells.
(a) Three different target sites (green) followed by the PAM sequence (red) in the Satb2 gene. (b) Schematic representation of the domain structure of Satb2. Arrowheads indicate the three sgRNA target sites used here. The anti-Satb2 antibody used in Figs 3 and 4 recognizes the C-terminal region of the Satb2 protein. (c) pX330-Satb2 plasmid and pCAG-EGxxFP-Satb2 target plasmid. pX330-Satb2 contains expression cassettes of humanized Cas9 and sgRNA for Satb2. pCAG-EGxxFP-Satb2 contains a genomic fragment including the sgRNA target sequence (black) between 5′ and 3′ EGFP fragments (green). (d) The effects of three kinds of pX330-Satb2 on EGFP expression derived from pCAG-EGxxFP-Satb2 target plasmids. pX330-Satb2, pCAG-EGxxFP-Satb2 and pCAG-mCherry were co-transfected into HEK293T cells. When pCAG-EGxxFP-Satb2 contained appropriate target sequences, HEK293T cells transfected with pX330-Satb2-272, -524 or -2129 exhibited EGFP signal in the majority of mCherry-positive transfected cells. pX330-Cetn1 and pCAG-EGxxFP-Cetn1 were used as positive controls. Scale bar = 200 μm. (e) The percentages of mCherry-positive transfected cells which became EGFP-positive. HEK293  cells were transfected with pX330-Satb2, pCAG-EGxxFP-Satb2 and pCAG-mCherry. N.S., not significant; one-way analysis of variance (n = 4 independent experiments). Error bars indicate SD.
