Cargando…

Genetic tools for the stable overexpression of circular RNAs

Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study g...

Descripción completa

Detalles Bibliográficos
Autores principales: Mecozzi, Nicol, Nenci, Arianna, Vera, Olga, Bok, Ilah, Falzone, Aimee, DeNicola, Gina M., Karreth, Florian A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8928841/
https://www.ncbi.nlm.nih.gov/pubmed/35289721
http://dx.doi.org/10.1080/15476286.2022.2043041
_version_ 1784670726323699712
author Mecozzi, Nicol
Nenci, Arianna
Vera, Olga
Bok, Ilah
Falzone, Aimee
DeNicola, Gina M.
Karreth, Florian A.
author_facet Mecozzi, Nicol
Nenci, Arianna
Vera, Olga
Bok, Ilah
Falzone, Aimee
DeNicola, Gina M.
Karreth, Florian A.
author_sort Mecozzi, Nicol
collection PubMed
description Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.
format Online
Article
Text
id pubmed-8928841
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher Taylor & Francis
record_format MEDLINE/PubMed
spelling pubmed-89288412022-03-18 Genetic tools for the stable overexpression of circular RNAs Mecozzi, Nicol Nenci, Arianna Vera, Olga Bok, Ilah Falzone, Aimee DeNicola, Gina M. Karreth, Florian A. RNA Biol Technical Paper Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo. We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs. Taylor & Francis 2022-03-15 /pmc/articles/PMC8928841/ /pubmed/35289721 http://dx.doi.org/10.1080/15476286.2022.2043041 Text en © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Technical Paper
Mecozzi, Nicol
Nenci, Arianna
Vera, Olga
Bok, Ilah
Falzone, Aimee
DeNicola, Gina M.
Karreth, Florian A.
Genetic tools for the stable overexpression of circular RNAs
title Genetic tools for the stable overexpression of circular RNAs
title_full Genetic tools for the stable overexpression of circular RNAs
title_fullStr Genetic tools for the stable overexpression of circular RNAs
title_full_unstemmed Genetic tools for the stable overexpression of circular RNAs
title_short Genetic tools for the stable overexpression of circular RNAs
title_sort genetic tools for the stable overexpression of circular rnas
topic Technical Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8928841/
https://www.ncbi.nlm.nih.gov/pubmed/35289721
http://dx.doi.org/10.1080/15476286.2022.2043041
work_keys_str_mv AT mecozzinicol genetictoolsforthestableoverexpressionofcircularrnas
AT nenciarianna genetictoolsforthestableoverexpressionofcircularrnas
AT veraolga genetictoolsforthestableoverexpressionofcircularrnas
AT bokilah genetictoolsforthestableoverexpressionofcircularrnas
AT falzoneaimee genetictoolsforthestableoverexpressionofcircularrnas
AT denicolaginam genetictoolsforthestableoverexpressionofcircularrnas
AT karrethfloriana genetictoolsforthestableoverexpressionofcircularrnas