PMC:4596894 / 9782-11056 JSONTXT

{"project":"2_test","denotations":[{"id":"26387595-21031596-2046276","span":{"begin":192,"end":194},"obj":"21031596"},{"id":"26387595-17460227-2046277","span":{"begin":334,"end":336},"obj":"17460227"}],"text":"Assays of Genetic and Functional Complementation\nWe performed genetic complementation of fibroblasts by transfecting the cells from HS individuals with PEX cDNA as described in Ebberink et al.19 To test the functionality of the PEX variants, we co-transfected pcDNA3-PEX1 or -PEX6 plasmids with the peroxisomal matrix marker pEGFP-SKL21 into skin fibroblasts deficient in PEX1 or PEX6. Cells transfected with only pEGFP-SKL served as negative controls, whereas co-transfections of the marker with pcDNA3 vectors containing the respective wild-type PEX cDNA served as positive controls. We subsequently analyzed the localization of the fluorescent signal 3 days after transfection by using the fluorescence microscope Zeiss Axio Observer A1.\nTo evaluate the effect of the variants found in the affected individuals, we determined per transfection the percentage of cells showing a punctate GFP signal (indicating “peroxisome-positive” or “complemented” cells) of the total number of 100–200 transfected cells. These ratios were normalized to the complementation efficiency of the positive control (set as 100%) and averaged per construct (n = 5–7). We used the one-sample Wilcoxon signed-rank test to test the statistical significance of deviations from the positive control."}