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In vivo and in vitro screening of cell-penetrating peptides based on CRE recombinase activity

Cell Penetrating peptides are small peptidic sequences that, upon fusion, allow the cellular uptake of cargo molecules bearing intracellular biological activity. The characterization of a growing number of peptides with potential CPP activity needs to develop quantitative procedures to assess their actual CPP activity. The present result is a screening procedure to evaluate the efficiency of Cell Penetrating Peptides (CPPs) as vectors for intracellular protein delivery. Intracellular delivery is monitored by the intracellular recombination activity of exogenously added CRE recombinase (the cargo) fused to the tested CPP (the vector).

In a dedicated engineered stable cell line, CRE-dependent recombination induces the permanent expression of a reporter protein (Beta-Galactosidase). The screen first consists in a 96 well format procedure allowing the accurate quantification of the intracellular delivery of CRE:CPP fusion protein in the reporter cell line. It is completed by a more physiologically relevant model based on embryonic brain explant culture from transgenic mice, to address the cellular tropism of the CRE:CPP fusion proteins.

The screening has been validated with different CRE:CPP fusion proteins and will be improved with small scale production of recombinant proteins.

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