Development of transgenic tools to assess adult stem cell potency and behavior in the regenerative flatworm Macrostomum lignano

Division Of Basic Sciences - Nci

Investigators

Abstract

We are devising and testing strategies for indelible labeling of M. lignano adult pluripotent stem cells and their progeny in in longitudinal studies using fluorescent microscopy. The ideal system would activate expression of a transgenic reporter in a stem cell, expression would be stable and inheritable by all progeny cells, and the activating event would be inducible. Several systems meet these requirements; we chose to develop the "CaSSA" and "STOP-lox" techniques in M. lignano because the enzymes used by these systems (Cas9 and Cre, respectively) can be activated directly by drug treatment. To maximize our chances of success, we are pursuing the development of both methods in parallel. Once a clone induction system is working as designed, we will shift our attention to its use for hypothesis-driven experiments. CaSSA, a Cas9-mediated recombination system for stable reporter expression in cell clones, relies on single-strand annealing (SSA) repair of double strand breaks by endogenous cellular machinery. CaSSA clonal labeling systems are in use in Drosophila, zebrafish, and mouse. To date, we have generated a CaSSA "switch" reporter construct and have demonstrated that recombination of this construct occurs when it is co-injected with Cas9 enzyme and sgRNA into zygotes. We generated a stable transgenic M. lignano line expressing a switch reporter, and a second line that expresses an 4-hydroxytamoxifen (4-OHT)-inducible Cas9-ERT2 fusion protein under the control of a ubiquitous promoter, along with sgRNAs driven by a U6 promoter. We are now conducting experiments to determine whether 4-OHT-inducible recombination occurs in F1 progeny of our mated transgenic lines, bearing one copy of the Cas9 transgene and one copy of the switch reporter. If 4-OHT does not promote Cas9-ERT2-mediated clone induction, we will test alternate drug-inducible Cas9 fusion proteins, including chemically inducible Cas9 (ciCas9) or DD-Cas9. In parallel, we are developing a "STOP-lox" inducible cell labeling system in which recombination is mediated by a drug-inducible Cre recombinase fused to ERT2. 4-OHT, an ERT2 ligand, is required for translocation of the Cre-ERT2 fusion protein to the nucleus, where it can catalyze recombination between loxP sites to effect production of a fluorescent reporter. We generated two stable transgenic lines required for this clone induction strategy, a ubiquitously expressed Cre-ERT2 transgene and a STOP-lox reporter, and are assaying for 4-OHT driven recombination of the reporter construct in vivo. If the that the Cre/loxP recombination does not work as expected in M. lignano and an alternative recombination system is required, we will pursue development of the CaSSA system (above) or a FLPase/FRT "FLP-out" clonal labeling system.