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Chapters
- 00:05Title
- 00:35Site-directed Mutagenesis
- 01:45Transformation and Screening for the Desired Clone
- 02:35pcDNA3.1/GLA and pcDNA3.1/GAA Plasmid Transfection and Treatment of HEK293H
- 04:00Cell Harvest
- 04:43Homogenization by Freezing and Thawing and BCA Assay
- 06:17Enzyme Activity Measurement with Artificial 4-Methylumbeliferyl Substrate
- 08:19Results: Enzyme Activity Measurement Mutagenized 3 α-galactosidase and 3 Acid α-glucosidase
- 09:35Conclusion
There is a demand to make pre-clinical testing for a novel class of "orphan" drugs called pharmacological chaperones reproducible, fast, and efficient. We developed a simple, highly standardized, and versatile cell culture-based assay to screen for eligible patients as well as novel pharmacological chaperone drugs.
Tags
In VitroEnzyme MeasurementPharmacological ChaperoneFabry DiseasePompe DiseasePhenotypic AssessmentAllelic VarianceLysosomal Storage DiseasesPrognostic OutcomesTherapeutic DecisionsSite-directed MutagenesisGLA GeneGAA GenePrimer DesignPCRDpn1E.coliPlasmid PurificationSpectrophotometerHEK293H CellsCell Transfection
