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TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex

Thiouracil (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...

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Autores principales: Tomorsky, Johanna, DeBlander, Leah, Kentros, Clifford G., Doe, Chris Q., Niell, Cristopher M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Society for Neuroscience 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659240/
https://www.ncbi.nlm.nih.gov/pubmed/29085897
http://dx.doi.org/10.1523/ENEURO.0181-17.2017
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author Tomorsky, Johanna
DeBlander, Leah
Kentros, Clifford G.
Doe, Chris Q.
Niell, Cristopher M.
author_facet Tomorsky, Johanna
DeBlander, Leah
Kentros, Clifford G.
Doe, Chris Q.
Niell, Cristopher M.
author_sort Tomorsky, Johanna
collection PubMed
description Thiouracil (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 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 (P)12, as well as from wild-type (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 BBB to label RNA in vivo.
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spelling pubmed-56592402017-10-30 TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex Tomorsky, Johanna DeBlander, Leah Kentros, Clifford G. Doe, Chris Q. Niell, Cristopher M. eNeuro New Research Thiouracil (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 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 (P)12, as well as from wild-type (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 BBB to label RNA in vivo. Society for Neuroscience 2017-10-04 /pmc/articles/PMC5659240/ /pubmed/29085897 http://dx.doi.org/10.1523/ENEURO.0181-17.2017 Text en Copyright © 2017 Tomorsky et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
spellingShingle New Research
Tomorsky, Johanna
DeBlander, Leah
Kentros, Clifford G.
Doe, Chris Q.
Niell, Cristopher M.
TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
title TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
title_full TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
title_fullStr TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
title_full_unstemmed TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
title_short TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex
title_sort tu-tagging: a method for identifying layer-enriched neuronal genes in developing mouse visual cortex
topic New Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659240/
https://www.ncbi.nlm.nih.gov/pubmed/29085897
http://dx.doi.org/10.1523/ENEURO.0181-17.2017
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