TU-Tagging is cell type-specific analysis of gene expression in vivo and in vitro that uses the uracil phosphoribosyltransferase (UPRT) gene of the protozoan parasite, Toxoplasma gondii, to convert the modified uracil 4-thiouracil into 4-thio-UMP for subsequent incorporation into mRNA.
Mike Cleary, Ph.D., assistant professor, University of California, Merced School of Medicine, explains that he developed this methodology as a graduate student. “You won’t find UPRT activity in multicellular animals, and thio-containing nucleotides don’t naturally occur in eukaryotic mRNAs, so selective purification of 4TU-tagged transcripts is possible.”
What Dr. Cleary found is that the thio-substituted compound 2,4-dithiouracil could be converted into a form that could connect to RNA. “If you start the pulse at 30 minutes, all RNA transcribed at that time will incorporate the TU Tag,” he explains. “I have shown that this works in UPRT-transgenic mouse models and can also be used to selectively tag T. gondii RNA during a mouse infection.”
Dr. Cleary also recently developed TU-tagging in a multicellular organism, the fruit fly, Drosophila melanogaster. He added that experiments in UPRT-transgenic flies “have demonstrated that TU-tagging can be used to purify cell type specific mRNAs under in vivo conditions with high sensitivity and specificity.”
Dr. Cleary recently received a California Institute of Medicine grant to further develop the TU-tagging technique by applying it to mammalian tissue culture cell lines and embryonic stem cell lines, and the mouse—two model systems that have proven useful for studying stem cell biology.
“As an example, this technique could allow stem cell-specific gene expression during interactions between stem cells and niche cells, analysis of gene expression in specific sub-populations of cells that arise during differentiation, identification of genes that are specifically expressed in stem, progenitor, and differentiated cells in vivo, and analysis of gene expression in cancer stem cells in vivo,” concludes Dr. Cleary. “TU-tagging could be an important part of the genomic toolkit. We’re still in the early phases, but there’s a good bit of preliminary data.”