Figure 2. Generation of BCAS2 conditional knockout (cKO) mice.

(a) Schematic representation of the BCAS2 gene targeting strategy. The LoxP-floxed allele contains one LoxP inserted between exons 2 and 3 and the other LoxP inserted between exons 4 and 5 with two Flip recombinase targets (FRTs) flanking the Neo cassette. The Neo cassette was removed by Flip recombinase. Exons 3 and 4 were deleted by CaMKIIα-iCre recombinase. CU and FD are primers for genotyping. (b) Genotyping by PCR. BCAS2^Flox/+;CaMKIIα-iCre⁺ offspring were crossed with each other, which resulted in 6 different genotypes. Genomic DNA was extracted from tails of offspring. Upper: The sizes of the bands identified by CU and FD primers were 0.8 kb for BCAS2^+/+, 0.8 and 0.7 kb for the heterozygous BCAS2-floxed allele (BCAS2^Flox/+), and 0.7 kb for the homozygous BCAS2-floxed allele (BCAS2^Flox/Flox). Lower: Cre gene. In this study, BCAS2^Flox/Flox;CaMKIIα-iCre⁺ mice were considered the forebrain-specific BCAS2 cKO mice and were named cKO; the WT mice were BCAS2^+/+ and contained the Cre gene. (c) RT-PCR analysis of the BCAS2 mRNA expression in WT and BCAS2 cKO mice at 12 weeks of age. Left, representative RNA. Right, quantification of the left panel (n = 3). (d) Western blot analysis of the BCAS2 protein levels for each genotype at 12 weeks of age. Left, representative western blot. Right, quantification of the left panel (n = 3). (e) Immunofluorescence assay (IFA) showing the BCAS2 expression level in 12-week-old WT and cKO mice. White arrow: BCAS2 expression in astrocytes of cKO mice. Scale bar: 200 μm. Uncropped blots are presented in Supplementary Fig. S5.