Abstract
TU-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4-thiouracil (4TU). This technique has been applied in mouse using a Cre-dependent UPRT transgene, CA > GFPstop > HA-UPRT, to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here we crossed the CA > GFPstop > HA-UPRT transgenic mouse to a Sepw1-cre line to express UPRT in layer 2/3 of visual cortex or to an Nr5a1-cre line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day 12, as well as from WT mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here we show that Cre-dependent UPRT expression can be used to purify cell type specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the blood brain barrier to label RNA in vivo.
Significance Statement Visual cortex has a laminar organization with neurons in each layer having distinct functional characteristics and developmental timelines. Previous studies looking for layer specific gene expression have examined embryonic, newborn or adult cortex, while overlooking significant post-natal stages. Here we used the transcriptional profiling technique, TU-tagging, to isolate transcripts enriched in visual cortex layer 2/3 at P12, a time of prolific synapse formation in this area. TU-tagging achieves spatial specificity via Cre-dependent expression of UPRT, and temporal specificity via subcutaneous injection of 4-thiouracil (4TU). This study is the first to successfully use a transgenic mouse to apply TU-tagging to neuronal cell types and to demonstrate that 4TU can cross the blood brain barrier to tag neuronal RNA.
Footnotes
The authors report no conflicts of interest.
This work was supported by NIH grant numbers T32-HD007348, and DP2-EY023190, and by HHMI, where CQD is an Investigator.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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