In situ hybridization with fluorochrome-labeled cloned DNA for quantitative determination of the homologous mRNA in individual cells.

Abstract

The transcriptional activity of genes may provide a reliable parameter for the ability of cells to synthesize and express gene products. In order to detect such transcription products not only qualitatively but also quantitatively, a method was developed to fluorochrome label cloned DNA coding for part of the constant fragment of the immunoglobulin mu-chain. Microfluorimetry of the bound fluorochrome allows a quantitative estimation of the amount of cloned DNA in the DNA-mRNA hybrids formed in the cytoplasm of individual cells by in situ hybridization and thus a quantitative estimation of the respective mRNA content. Moreover, upon appropriate preparation of the cells the simultaneous quantitative detection of surface antigenic properties with antibodies labeled with a second fluorochrome is possible. Such a cell preparation procedure was optimized according to the highest signal obtainable. In the system chosen, exemplary cells from normal donors and different lymphoproliferative disorders were investigated for their ability to express the immunoglobulin mu-chain. Whereas a B-CLL was positive for the mu-mRNA and a T-CLL was negative for it as expected, a number of non-T non-B cALLs contained varying fractions of mu-mRNA positive cells with varying intensities. This method will allow a more exact definition of differentiative steps in B-cell development with respect to surface antigenic pattern, activation of the immunoglobulin genes, first of all the mu-gene, and immunoglobulin content and expression. The same method can also be applied to other gene sequences for which cloned DNA fragments or cDNA is available, and for which the transcriptional activity in cells of defined surface antigenic properties is to be determined.