Fig. 1

Generation and characterization of mice lacking glycosaminoglycan chains from perlecan core protein. A) Perlecan primary structure. Serine residues (S, asterisks) in domains I and V are predicted attachment sites for glycosaminoglycan (GAG) chains. Exons 3 and 91 (red boxes) were removed from the perlecan gene (Hspg2) and exon 3 was replaced with a neo cassette (grey box). Black triangles, loxP sequences. B) Genotyping of Hspg2^+/+, Hspg2^+/- and Hspg2^∆3∆91 mice. C) Primers from the targeted exons 3 and 91 showed no qPCR amplicon in the Hspg2^∆3∆91 mice, indicating the absence of GAG attachment sites. qPCR using primers from exons 2, 4 or 94 showed no differences between the genotypes, demonstrating unaltered Hspg2 mRNA expression (n = 5 mice per genotype). D) Brain homogenates were treated with heparinase (+) or left untreated (-) and analysed by western blot using anti-perlecan antibody. In the control samples (Hspg2^+/+ and Matrigel^®) perlecan is visible in western blot only after removal of the GAGs (heparinase +). Perlecan core protein is readily detectable in the Hspg2^∆3∆91 mice without heparinase, and the treatment does not affect perlecan mobility, indicating the absence of GAGs in the Hspg2^∆3∆91 protein. Vinculin was used as a loading control. E) Immunofluorescence staining of perlecan core protein (red) and Col IV (green). Perlecan is located in the perivascular BMs of the Hspg2^+/+ and Hspg2^∆3∆91 mice, quantification of the data in F and G (n = 5–6 per genotype). There is no perlecan immunoreactivity in the mice devoid of perlecan core protein (Hspg2^-/-). H) qPCR of the brain hemisphere shows no differences in the expression level of basement membrane components in Hspg2^∆3∆91 (n = 5) mice relative to Hspg2^+/+ (n = 5). Scale bar, 200 μm (E). The statistical tests used were the multiple unpaired two-tailed t-test with Welch’s correction followed by Benjamini and Hochberg correction for multiple tests (C, H), the Mann-Whitney unpaired two-tailed t-test (F) and the unpaired two-tailed t-test with Welch’s correction (G). ***p < 0.001, ****p < 0.0001. ns, non-significant. Mean ± SEM