Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka

Abstract

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

Small-Molecule Modulation of Protein Homeostasis

“…The Shield system has been successfully employed in a wide range of organisms including zebrafish, transgenic medaka, Plasmodium falciparum , , and Toxoplasma gondii but has proved not to be amenable to use in Saccharomyces cerevisiae …”

“…The Shield system can also be incorporated into specific tissues in transgenic mice via the CRE/Lox approach, thereby providing a dual-inducible system. 180 The Shield system has been successfully employed in a wide range of organisms including zebrafish, 181 transgenic medaka, 182 Plasmodium falciparum, 183,184 and Toxoplasma gondii 185 but has proved not to be amenable to use in Saccharomyces cerevisiae. 186 TALEN and CRISPR/Cas9 have been employed to knock in the shield system destabilization domain into cancer targets (p53 and PI3Kα).…”

Targeted Protein Degradation by Small Molecules

“…This system makes used of a destabilized mutant of FKBP12 that is stabilized in the presence of the compound Shld-1 and has proved useful in controlling fusion protein stability in parasites (95), worms (96), and medaka (97) as well as to potassium channel biology (98) and the cytosolic UPR in mammalian cells (99). Several technological advancements are worth noting: By combining Shld-1 stabilization with induced dimerization of a split-ubiquitin system, release of the native protein is achieved in response to a small molecule (100).…”