Institut for Biomedicin

Lars Bolund

Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells

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  • Institut for Human Genetik
  • Klinisk Genetisk Afdeling
  • Immunologi og mikrobiologi
  • HBS, Sygdomsmekanismer, -markører og -forebyggelse
  • Miljø- og Klimateknik
  • Ortopædkirurgisk Afdeling E, THG
  • Molekylær Medicinsk Forsk.enhed
To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cell surface is quantitated. The receptor activity is measured by incubating the cells with fluorescence labeled LDL (Dil-labelled LDL) at 37 degrees C and 4 degrees C. The transfected cells stained with anti-LDL-R antibodies can also be analysed by immunofluorescence microscopy allowing the study of the intracellular location of variants of the receptor. To evaluate these methods, we are analyzing four previously well-characterized LDL-R mutations, belonging to each of the classes 2 to 5. Preliminary data show that mutant genes belonging to class 3 and 4A give rise to receptor protein on the cell surface, but impaired LDL uptake, while mutant receptors belonging to class 2A and 5 can only be detected intracellularly. Expression of the class 2A mutation results in an ER staining pattern, whereas the class 5 mutation gives rise to an intracellular staining compatible with localization in the endosomal/lysosomal compartments. We conclude that this system is useful for a rapid functional analysis of newly discovered mutations in the LDL-R gene.
OriginalsprogEngelsk
TidsskriftZeitschrift fuer Gastroenterologie
Vol/bind34 Suppl 3
Sider (fra-til)9-11
Antal sider3
ISSN0044-2771
StatusUdgivet - 1 jun. 1996

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