Xenopus transgenic to screen candidate molecules favoring myelin repair

摘要

To facilitate live imaging of demyelination and remyelination, we have generated a Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelinating oligodendrocytes. In this line, the proximal portion of mouse myelin basic protein (MBP) regulatory sequence, specific to mature myelin-forming oligodendrocytes, drives the expression of a transgene protein formed by the GFP reporter fused to the Escherichia coli nitroreductase (NTR) selection enzyme. The NTR enzyme converts the innocuous prodrug metronidazole (MTZ) to a cytotoxin. The demyelination response of MBP-GFP-NTR tadpoles to MTZ is followed by spontaneous remyelination after cessation of MTZ treatment. Thanks to the transparency of tadpoles, these events can be monitored in vivo by two-photon imaging and easily quantified on a simple fluorescence macroscope. We have used the MBP-GFP-NTR model to screen in vivo molecules favoring remyelination. At the end of MTZ-induced demyelination, tadpoles were switched to water containing the compounds to be tested. After 3 days of treatment remyelination was assayed by counting the number of GFP+ oligodendrocytes per optic nerve. Using Crispr/Cas9 strategy, the target of the candidate molecule can be easily deleted in the MBPGFP-NTR line to examine the mechanism of action of the candidate molecule. Therefore the Xenopus laevis transgenic line, MBP-GFP-NTR, allowing conditional ablation of myelinating oligodendrocytes, constitutes a new medium-throughput screening platform for myelin repair therapeutics in demyelinating diseases.