Developmental and tissue specific regulation of globin gene expression has been the subject of intense investigation as part of an effort to develop a general model of eukaryotic gene regulation. In addition, pharmacologic manipulation of globin gene expression in human erythroid cells holds considerable promise as a means of controlling the clinical course of thalassemia and sickle cell disease. A system for assay of gene expression in single cells is being developed in order to elucidate the mechanism by which pharmacologic agents modulate the level of expression of globin gene. Owing to the limited quantity of mRNA contained within a single cell, the assay involves amplification of poly (A) RNA in a template independent manner while maintaining their relative abundance. This involves reverse transcription of 500-700 bases from the 3' end of each mRNA followed by poly (A) tailing of the 3' end of the new cDNA and PCR amplification using oligo(dT) based primers. Assay of specific templates by PCR coupled with real time detection of a fluorescence signal produced from a labeled gene specific probe provides the quantitation. The assay system is currently being tested using a K-562 cell line which expresses human fetal and embryonic globins. A considerable amount of heterogeneity of embryonic globin expression has already been observed between cells using agarose gel electrophoresis and ethidium bromide detection of amplified products; the implementation of real time detection will allow for rigorous quantitation of the initial amount of template. It is anticipated that such a quantitative approach will ultimately permit the identification of candidate genes as targets of drug action and contribute to a greater understanding of the globin gene switching phenomenon.
