Skip to main content
. 2023 Jun 22;29(12):3786–3801. doi: 10.1111/cns.14299

FIGURE 2.

FIGURE 2

Generation of GCPII, GCPIII, and GCPII/III gene knockout mice by CRISPR/Cas9‐mediated genome engineering. (A) Schematic of the strategy used to generate GCPII, GCPIII, and GCPII/III knockout mice. (B, C) Genotyping of GCPII, GCPIII, and GCPII/III knockout mice by RT–PCR analysis of tail DNA samples. (B) The molecular weight of the PCR product of GCPII was 1605 bp in wild‐type mice and GCPIII−/− mice and 808 bp in GCPII−/− and GCPII/III−/− mice (GCPII, P3 and P4 primers), while the molecular weight of the PCR product of GCPIII was 3796 bp in wild‐type and GCPII−/− mice and 758 bp in GCPII−/− and GCPII/III−/− mice (GCPIII, P3 and P4 primers). (C) The molecular weight of the PCR product of GCPII was 405 bp in wild‐type and GCPIII−/− mice (GCPII, P1 and P2 primers). No band was detected in GCPII−/− and GCPII/III−/− mice. The molecular weight of the PCR product of GCPIII was 423 bp in wild‐type mice and GCPII−/− mice (GCPIII, P1 and P2 primers). No band was detected in GCPIII−/− and GCPII/III−/− mice. (D) RT–PCR analysis of GCPII and GCPIII expression in the hippocampus of wild‐type, GCPII−/−, GCPIII−/−, and GCPII/III−/− mice. The molecular weight of the PCR product of GCPII was 405 bp in wild‐type and GCPIII−/− mice (GCPII, P1 and P2 primers). No band was detected in GCPII−/− and GCPII/III−/− mice. The molecular weight of the PCR product of GCPIII was 423 bp in wild‐type mice and GCPII−/− mice. No band was detected in GCPIII−/− and GCPII/III−/− mice (GCPIII, P1 and P2 primers). Gapdh was used as an endogenous reference gene. (E) Western blot analyses of GCPII expression in the hippocampus of wild‐type, GCPII−/−, GCPIII−/−, and GCPII/III−/− mice. A band over 100 kDa was detected in the wild‐type and GCPIII−/− mice, while no GCPII protein was detected in GCPII−/− and GCPII/III−/− mice. β‐Actin was used to control for loading. (F) Immunofluorescence, GCPII (green) in the hippocampus of wild‐type, GCPII−/−, GCPIII−/−, and GCPII/III−/− mice. Scale bar = 50 μm. (G) The expression levels of NEUN, GFAP, TNF‐α, and IBA1 in hippocampus of wild‐type, GCPII−/−, GCPIII−/−, and GCPII/III−/− mice were detected by immunoblotting, and β‐Actin was used to control for loading. The right panel (H) is the quantitative analysis of the protein bands (n = 3 per group, one‐way ANOVA, p > 0.05 among all groups). The errors bars indicate the S.E.