Journal
RNA BIOLOGY
Volume 19, Issue 1, Pages 353-363Publisher
TAYLOR & FRANCIS INC
DOI: 10.1080/15476286.2022.2043041
Keywords
Circular RNA; transposon; melanoma; mouse model; ESC-GEMM; sleeping beauty; piggyBac
Categories
Funding
- National Cancer Institute of the National Institutes of Health [R37CA230042, R03CA227349, R01CA259046]
- Melanoma Research Alliance [MRA Young Investigator Award]
- Gene Targeting Core - Moffitt's Cancer Center Support Grant from the National Cancer Institute of the National Institutes of Health [P30CA076292]
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Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in biological processes. However, studying the functions of circRNAs can be challenging due to limitations of traditional approaches. This study developed genetic tools for stable circRNA overexpression and demonstrated their applications in vitro and in vivo.
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.
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