The thio-RNA is then in vitro-biotinylated, purified from total RNA, and used for gene expression analyses via next-generation Mice, thereby preserving normal cell interactions and organismal physiology during the window of RNA labeling ( Fig. Importantly, production of the thio-RNA occurs within the intact tissue in living Only the cell types expressing UPRT will efficiently incorporate 4TU into newly transcribed RNA, thereby covalently Temporal specificity is via injection of the uracil analog 4-thiouracil (4TU) ( Fig. Uracil phosphoribosyltransferase (UPRT) ( Fig. Spatial specificity is obtained by Cre-induced expression of a transgene encoding In specific cell types within intact mice. TU tagging is a genetic and chemical intersectional approach that allows covalent labeling of actively transcribed RNAs 2009) and pioneering work in cell culture ( Cleary et al. Here we describe a method called mouse thiouracil (TU) tagging that is based on our previous work in Drosophila ( Miller et al. Of cellular RNA (messenger RNA or microRNA ), each requires overexpression of an endogenous mouse protein thatĬould have deleterious effects, and each provides only limited temporal control of labeling. These methods succeed in avoiding dissociation trauma, but they, too, have limitations. Intact tissues without the need for cell dissociation ( Heiman et al. To these problems, several genetic methods have been recently developed for isolating RNA from specific cell types from within Mechanical purification of epithelial cells may thus alter gene expression prior to transcriptional profiling. That act as signaling “hubs,” and loss of junctional integrity triggers gene expression changes ( Balda and Matter 2009 Stepniak et al. For example, epithelial cells have apical junctions Nonphysiological changes in gene expression during the dissociation procedure. Methods run the risk of losing RNA in fine cellular processes (e.g., axons, dendrites, or glial processes) and can induce ![]() In practice, many of these methodsĪre slow or laborious, require expensive equipment, or are not capable of isolating dispersed cell types. While these methods are effective, they have practical and theoretical limitations. The most common methods for isolating cells for transcriptional profiling-fluorescence-activated cell sorting (FACS), laserĬapture, manual dissection, and panning-require dissociation of the targeted cells from their host tissue ( Lobo et al. Within its native environment (e.g., before and after differentiation, cell migration, host/bacterial interactions, or neuronal It has proven difficult, however, to monitor acute gene expression changes in a specific cell type Provide insights into how cell types develop and function, communicate with each other, contribute to disease, and are affectedīy therapeutic treatments. Transcriptional profiling in mouse models can The mammalian body is composed of a complex assembly of distinct cell types. Transcribed genes in specific cells at specific times within intact mice. TU tagging provides a novel method for identifying actively Moreover, generating chimeric mice via UPRT + bone marrow transplants identifies immune versus niche spleen RNA. Induced by lipopolysaccharide (LPS) injection. This method can purify transcriptsįrom spatially complex and rare (<5%) cells, such as Tie2:Cre + brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely Only UPRT + cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). Injection of 4-thiouracil (4TU) provides temporal specificity. ![]() Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity We describe “mouse thiouracil (TU) tagging,” a genetic and chemical intersectional method for covalent labeling and purification ![]() Purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Transcriptional profiling is a powerful approach for understanding development and disease.
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