Long-Term Labeling and Differentiation Monitoring of Mouse Spermatogonial Stem Cells Using CellREADR
TL;DR
This work identified several previously unreported features of CellREADR, including RNA interference induced by double-strand RNA formation that is obligatorily generated during the normal operation of the CellREADR system, and established its application for long-term labeling of mSSCs and monitoring mSSC differentiation induced by retinoic acid treatment in vitro.
Abstract
Cellular identity determination and lineage tracing is a pivotal technique in modern biological research. Conceptually simple yet efficient cell labeling techniques offering broader applicability are warranted despite many methods requiring intricate construction procedures. One such technique is ADAR (adenosine deaminase acting on RNA)-mediated RNA sensing, a live cell labeling technique that is based on the expression and abundance of cell-type-specific RNAs. Here, we utilized the optimized version, CellREADR (Cell access through RNA sensing by Endogenous ADAR), to establish a feasible tracing system for mouse spermatogonial stem cells (mSSCs) which are refractory to CRISPR-based reporter gene knock-in. We identified several previously unreported features of CellREADR, including RNA interference induced by double-strand RNA formation that is obligatorily generated during the normal operation of the CellREADR system. More importantly, we established its application for long-term labeling of mSSCs and monitoring mSSC differentiation induced by retinoic acid (RA) treatment in vitro. This work offers a practical solution for dynamic monitoring of mSSC self-renewal and differentiation and supports that CellREADR can be developed into more versatile and efficient tools in stem cell research.