Creating conditional dual fluorescence labeled transgenic animals for studying function of small noncoding RNAs

ABSTRACT

Because the function of most noncoding (nc) RNAs is unknown, Cre-lox transgenic mice are useful tools to determine their functions in a tissue or developmental stage-specific manner. However, the technology faces challenges because expression of ncRNA-transgene lacks protein product. No antibody or peptide-tag can be used to trace ncRNA expression in mouse tissues in real time. Furthermore, transgene integration at different locus or orientations in the genome may result in recombination of genomic fragments in the Cre-lox system. Establishing a reliable method that can be used to determine the precise copy number and orientation of the transgene is critical to the field. We developed a fast and straightforward method to determine ncRNA-transgene copy number, orientation, and insertion site in the genome. Furthermore, upon tissue-specific expression of ncRNA, a Cre-loxP-mediated dual-fluorescence expression system facilitates fluorescence signal switching from green to red, which enables real-time monitoring of ncRNA expression by fluorescence signals. As proof of concept, we demonstrate that after microRNA (miRNA)-Flox mice crossed with Col2a1-Cre mice, miRNA transgene expression could be detected successfully by red fluorescence signals in various cartilaginous tissues. This method of creating small ncRNA transgenic mice facilitates both tissue-specific ncRNA expression and real-time visualization of its expression. It is particularly suitable for in vivo studies of the functional roles and lineage tracing of small ncRNA.

KEYWORDS:

• Droplet digital PCR
• dual fluorescence switching system
• microRNA
• noncoding RNA
• transgene copy number
• transgenic mice

Acknowledgment

We thank Dr. Wentian Yang for the critical reading of this paper and providing Col2a1-cre mice.

Funding

This study was supported by grants from the National Institutes of Health (P20GM104937 to Q.C., and 1R21NS095635 and 1R21NS092127 to Z.X.).

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.

Additional information

Funding

This study was supported by grants from the National Institutes of Health (P20GM104937 to Q.C., and 1R21NS095635 and 1R21NS092127 to Z.X.).