Single Cell Analysis of Cre-based Activation of Global Transcription

The organs of eukaryotic organisms in general comprise a complex interspersion of different cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease states, particularly oncogenesis, involves alterations in the genetic and epigenetic state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease states within the overwhelming background of normal cells. We have generated a number of transgenic mouse lines expressing a nuclearly-localized version of the Green Fluorescent Protein (GFP), in which production of a chimeric histone 2B-GFP protein is under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny, excision of the STOP sequence activates transcription and is identified by the emergence of green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei. We describe strategies for employing these lines in the transcriptomic analysis of oncogenesis.