Inducible Cassette Exchange: A Rapid and Efficient System Enabling Conditional Gene Expression in Embryonic Stem and Primary Cells

TLDR

Genetic modification is essential for studying cell fate specification and maintenance, yet current engineering methods are inefficient. The study demonstrates a rapid, efficient inducible cassette exchange system that replaces a floxed Cre allele with an incoming transgene and illustrates its utility by inserting the myogenic regulator Myf5 into the ICE locus. The ICE system targets a floxed Cre allele at the HPRT locus, enabling recombination in murine embryonic stem cells and primary cells, and, via lentivectors, achieves recombination at a randomly integrated ICE locus in human ESCs. The system enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells, making it a versatile tool for gain‑of‑function studies on gene function in cell fate specification and reprogramming.

Abstract

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.