Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells.
Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.
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Department of Molecular Immunology, Genentech Inc., South San Francisco, California 94080.
Pennica
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D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics*; D002459:Cell Fusion; D002461:Cell Line, Transformed; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005786:Gene Expression Regulation; D006801:Humans; D013861:Thiocyanates; D014162:Transfection
D000945:Antigens, Differentiation, T-Lymphocyte; D005452:Fluoresceins; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate