Measurement of transient cDNA expression in mammalian cells using flow cytometric cell analysis and sorting.
Generalized methods for quantitative and sensitive measurement of transient cDNA expression in mammalian cells using flow cytometry (FCM) are described. The techniques are applicable to a wide variety of cDNAs encoding intracellular or cell surface protein products through the use of immunofluorescence- or nonimmunofluorescence-based detection methods. The methods illustrated have been optimized for sensitive detection of transfectants and efficient recovery of the encoding plasmids from the sorted cells. Expression levels and heterogeneities were compared using four methods of DNA transfer in addition to description of a novel method to optimize single copy transfer probabilities by multiparameter analysis. The overall sensitivities are compared by reconstruction and molecular cloning experiments to other methods of selection, such as immunoselection by panning. Through the measurement of multiple heterologous products per cell, or the measurement of multiple epitopes or binding sites per heterologous protein, expression levels on a single cell basis can be measured and correlated with other endpoints for various purposes. The ability to detect and recover rare clones based on a number of single and multiparameter selection criteria should significantly extend the use of transient mammalian cDNA expression methods for applications involving novel FCM-based reporter cDNA assays and for cloning certain rare surface-bound or secreted proteins using FCM.
Rice
G C
GC
Genentech, Inc., So. San Francisco, California 94080.
Pennica
D
D
Borree
J A
JA
Williams
S R
SR
D000818:Animals; D002469:Cell Separation / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D003637:DEAE-Dextran / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D014162:Transfection
D004247:DNA; D003637:DEAE-Dextran