Microanalysis of gene expression in cultured cells
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In this thesis two aspects of gene expression in cultured cells have been studied: the heterogeneity in gene expression in relation with the development and application of microchemical techniques for the prenatal diagnosis of inborn errors of metabolism and the possibility of inducing gene expression in cultured cells using somatic cell hybridization. The (prenatal) diagnosis of genetic metabolic diseases is based on a comparison of data after biochemical analysis of cultured amniotic fluid cells and fibroblasts from normal individuals, heterozygotes and affected patients or fetuses. For a reliable prenatal diagnosis the effect of cell cultivation conditions and various morphological cell types within one culture on the biochemical parameters to be studied must be known. In this thesis the activity of three lysosomal enzymes, acid a-glucosidase, S-galactosidase and S-N-acetylhexosaminidase were investigated in relation to different cell types in primary amniotic fluid cell cultures. This was possible by microchemical analysis on small groups (10- 100) of morphologically different clones isolated from freeze-dried amniotic fluid cell cultures. The activities of the three lysosomal enzymes when expressed per cell appeared to be about the same in the small epithelioid cell types 1 and 2 and the fibroblast-! ike cells. In the large, often multinuclear cells however, the enzyme activity per cell was 5 to 10 fold higher than this in the other cell types. By performing combined microinterferometric determinations of the dry mass and enzyme assays on single cells we were able to show that no differences in lysosomal enzyme activities exist between the various amniotic fluid cell types if the activity is expressed per unit dry mass (appendix paper 1).
Het onderzoek werd mede mogelijk gemaakt door financie1e steun van het Preventiefonds, Den Haag.
- chick erythrocytes
- fluid cells
- cell types