Thiouracil (TU) tagging is a method developed for isolating RNA from specific cell types from within intact tissues without the need for cell dissociation.
In this transgenic strain, a ubiquitous chicken β-actin-CMV (CAG) promoter drives expression of a loxP-GFP-3xstop-loxP cassette followed by a hemagglutinin (HA) epitope-tagged uracil phosphoribosyltransferase (UPRT) gene. Embryonic day 12.5 (E12.5) embryos and postnatal day 6 (P6) floxed animals show a widespread pattern of GFP fluorescence. The GFP-stop cassette effectively prevents any leaky read-through of UPRT expression from the floxed allele. Cre-induced excision of the floxed GFP-stop permits tissue-specific spatial expression of UPRT. Subsequent subcutaneous injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT+ cells exposed to 4TU produce thio-RNA which can then be purified for gene expression analysis via next-generation RNA sequencing (RNA-seq). TU tagging has been shown to have neglible effect on gene expression in cell lines and ubiquitous expression of UPRT has no effect on viability in mice.
When crossed with Tie2-cre (Tek-cre) animals, double-transgenic six-day-old mice show robust UPRT expression in PECAM+ (CD31) endothelial cells of the cerebellum and all other regions of the brain (e.g cortex, dentate gyrus, midbrain, choroid plexus, and hypothalamus) as well as Tie2-cre-expressing endothelial cells of the heart. Expression of UPRT is also seen in embryonic day 11.5 (E11.5) brain and heart.
When the floxed interval is excised by Math1-cre (Atoh-cre), mice show robust expression of UPRT in cerebellar granule neuron precursors (GNPs) of the postnatal day 6 brain.
Transplantation of UPRT+ cells can be carried out to create chimeric mice, useful in hematopoietic studies of cell migration and morphogenesis. TU tagging is complementary to cell isolation methods such as FACS panning and laser capture, with the added refinement of isolating only nascent RNA.
