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Figure 1 | Molecular Medicine

Figure 1

From: Induction of Tolerance to Human Arylsulfatase A in a Mouse Model of Metachromatic Leukodystrophy

Figure 1

In vitro mutagenesis of the wildtype hASA cDNA and expression of hASA-C69S in cultured cells. (A) Exchange of three nucleotides (underlined) of the wildtype hASA cDNA resulted in the substitution of cysteine by serine at position 69 (bold letters), the loss of an ApaLI restriction site and the gain of a ScaI site. (B) The hASA-C69S cDNA was cloned under the control of the chicken β-actin promoter of the eukaryotic expression vector pTVC. (C) BHK cells were transiently transfected with pTVC harboring the hASA-C69S in 5′–3′ or, as a control, in 3′–5′ orientation. For additional controls, pBEH-HT14/CP8 bearing the wildtype hASA cDNA under the control of the SV40 promoter (15), the empty TVC plasmid (pTVC) or no DNA was used. ASA was quantified by ELISA (upper panel) or activity assays (lower panel) using cell homogenates (bars) and conditioned medium (closed circles). (D) Immunofluorescence of wildtype (wt) and mutant hASA (C69S) in BHK cells transiently transfected with pBEH-HT14/CP8 and pTVC hASA-C69S 5′–3′, respectively. The red perinuclear hASA staining of the original color images is visible as a bright signal after conversion into the black and white image. The DAPI staining of the nuclei appears gray.

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