Figure 2.

Decreased SORLA expression in the brain of SORL1-deficient Göttingen minipigs

(A) RT-PCR validation of SORL1-depleted Göttingen minipigs. cDNA obtained from reverse transcription of total RNA isolated from the cerebellum (Cb) or frontal cortex (Cx) from wt, het, and ko SORL1 Göttingen minipigs was used as a template in RT-PCR using primers specific for SORL1 exons 1 and 2 (see primers F1+R2 in Figure 1D), SORL1 exons 46 and 47 of the SORL1 -202 transcript, or for porcine GAPDH (reference gene). Negative controls (ctrl) correspond to reactions carried out in parallel using −RT) or with H₂O as templates.

(B and C) Relative SORL1 mRNA expression in SORL1 het (n = 4) minipigs was measured by qPCR, calculated relative to the reference gene HPRT1, and normalized to SORL1 expression in SORL1 wt minipigs (n = 4) for samples from the Cb (B) and Cx (C).

(D) WB analysis confirming complete absence of SORLA expression in Cb or frontal Cx homogenates from the SORL1 ko compared with a wt and a het SORL1 Göttingen minipig (D).

(E) Homogenates of Cx from het and wt SORL1 minipigs were analyzed by WB to determine expression levels of SORLA, APP, VPS26B, and VPS26A, using the level of ACTIN as a loading ctrl.

(F–I) The expression levels of SORLA (F), APP (G), VPS26B (H), and VPS26A (I) were quantified relative to the level of ACTIN.

(J) Homogenates of Cb from het and wt SORL1 minipigs were analyzed by WB to determine expression levels of SORLA, APP, and VPS26B, using the level of ACTIN as a loading ctrl.

(K–M) The expression levels of SORLA (K), APP (L), and VPS26B (M) were quantified relative to the level of ACTIN.

(N and O) Equal amounts of CSF from het and wt SORL1 minipigs (n = 4 in both groups) were immunoprecipitated and analyzed by WB using an antibody for SORLA detection (N), and the relative levels of sSORLA were quantified (O).

Identification numbers of individual minipigs are provided below every lane for all WB analyses. Two-tailed unpaired Student’s t test was used for all statistical analyses, with p values below 0.05 considered significantly changed. Data are expressed as mean ± SEM.