Coexpression of γ and β Globin mRNA in Cells Containing a Single Human β Globin Locus: Results From Studies Using Single-Cell Reverse Transcription Polymerase Chain Reaction

Abstract

AbstractWe developed a method detecting globin gene expression in single cells using reverse transcription polymerase chain reaction, ϵ and γ globin cDNAs are coamplified by an ϵγ primer set whereas γ and γ globin cDNAs are coamplified by a γβ primer set and the individual globin cDNAs are distinguished by restriction enzyme digestion. Analysis of RNA preparations from human fetal liver, neonatal red blood cells (RBCs), or adult RBCs showed the expected mRNA species for each stage of human development. Analysis of single cells from a human erythroleukemia line coexpressing γ and β globin chains showed heterogeneity in γ and β mRNA contents. The method was subsequently used to test whether only one or more than one globin genes are expressed in cells that contain a single human β globin locus. We found that about 50% of single cells from MEL × fetal erythroid cell hybrids containing a single human β globin locus coexpressed γ and β globin mRNA. This finding is best explained by assuming that both γ and β genes are simultaneously transcribed from the same β globin locus implying that the LCR can simultaneously interact with more than one globin gene promoter.