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bioRxiv · 10.1101/2025.05.23.655316

CellCousin2: An Optimized System for Partial Ablation and Tracing of Regenerative Lineages

Hovhannisyan, G. G.·Akhourbi, T.·Eski, S. E.·Pirson, I.·Gurzov, E. N.·Singh, S. P.
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Abstract

Regeneration can rely on multiple cellular sources, including stem cells, self-duplicating cells, and transdifferentiating cells. A central question in regenerative biology is how these distinct lineages contribute to repair and interact within a functional regenerate. We previously developed the CellCousin system to study hcellular plasticity using inducible recombination and nitroreductase-mediated ablation in zebrafish. Here, we present CellCousin2, which introduces two key improvements for long-term tracking of spared and regenerating cells. First, to reduce background recombination, we developed a Dihydrofolate Reductase (DHFR)-CreER system with dual control: DHFR- mediated degradation in the absence of trimethoprim, and tamoxifen-dependent activation. This combination minimizes leakiness while maintaining high recombination efficiency. Second, we replaced the original nitroreductase with NTR2.0, enabling effective ablation with tenfold lower metronidazole concentration, reducing off-target effects on the liver. Together, these enhancements make CellCousin2 a robust platform for dissecting the dynamics and interactions of regenerative lineages.

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Hovhannisyan, G. G., Akhourbi, T., Eski, S. E., Pirson, I., Gurzov, E. N., Singh, S. P.. 2025-05-28. CellCousin2: An Optimized System for Partial Ablation and Tracing of Regenerative Lineages. https://doi.org/10.1101/2025.05.23.655316

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