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Identifier

000380706

Title

Targeted cell ablation towards the identification of cell populations that participate in regeneration

Alternative Title

Ταυτοποίηση κυτταρικών πληθυσμών που συμμετέχουν στην αναγέννηση των άκρων μέσω επαγωγής κυτταρικού θανάτου

Author

Κολτσάκη, Ιωάννα

Thesis advisor

Αβέρωφ, Μιχαήλ

Abstract

Conditional targeted cell ablation is a powerful tool for determining the role of specific cell types in development and regeneration. A recently established chemical-genetic system based on the bacterial E.coli enzyme nitroreductase (NTR) is efficient for conditional cell ablation. NTR reduces the drug-substrate Metronidazole (Mtz) and converts it into a cytotoxic DNA-cross linking metabolite resulting in cell death. The NTR/Mtz cell ablation system was previously shown to function in zebrafish and mice, indicating that it is likely to be an effective tool in a wide range of animals. Ι established this system¬¬¬ in the amphipod crustacean Parhyale hawaiensis, which is an attractive model for studying regeneration, with a range of genetic tools based on transgenesis. Using a cell type specific promoter upstream of cfp-ntr gene (NTR fused to the Cyan Fluorescent Protein) and inserting it into Parhyale genome via Minos transposable element, I achieved to express the fused protein in specific cells and visualize its expression in the transgenic animals by the presence of CFP fluorescent marker. At low concentrations of Mtz substrate (which are otherwise harmless), I could ablate muscle cells and monitor the process through changes in CFP fluorescence. I tested FRT/Flipase (Flp) and Cre/lox recombination system in Parhyale by an excision assay. Such recombinase system will enable us to restrict NTR expression within clones of cells in specific cell types or tissues. By combining nitroreductase-mediated ablation and time-lapse imaging, we will be able to monitor regenerating legs after the ablation of specific cell types (e.g. muscle or neural cells) to determine their role in limb regeneration.

Language

English

Issue date

2012-11-16

Collection

School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses

Type of Work--Post-graduate theses

Views

272

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