Efficient CPP-mediated Cre protein delivery to developing and adult CNS tissues.
BACKGROUND
Understanding and manipulating gene function in physiological conditions is a major objective for both fundamental and applied research. In contrast to other experimental settings, which use either purely genetic or gene delivery (viral or non-viral) strategies, we report here a strategy based on direct protein delivery to central nervous system (CNS) tissues. We fused Cre recombinase with cell-penetrating peptides and analyzed the intracellular biological activity of the resulting chimerical proteins when delivered into cells endowed with Cre-mediated reporter gene expression.


RESULTS
We show that active Cre enzymatic conjugates are readily internalized and exert their enzymatic activity in the nucleus of adherent cultured cells. We then evaluated this strategy in organotypic cultures of neural tissue explants derived from reporter mice carrying reporter "floxed" alleles. The efficacy of two protocols was compared on explants, either by direct addition of an overlying drop of protein conjugate or by implantation of conjugate-coated beads. In both cases, delivery of Cre recombinase resulted in genomic recombination that, with the bead protocol, was restricted to discrete areas of embryonic and adult neural tissues. Furthermore, delivery to adult brain tissue resulted in the transduction of mature postmitotic populations of neurons.


CONCLUSIONS
We provide tools for the spatially restricted genetic modification of cells in explant culture. This strategy allows to study lineage, migration, differentiation and death of neural cells. As a proof-of-concept applied to CNS tissue, direct delivery of Cre recombinase enabled the selective elimination of an interneuron subpopulation of the spinal cord, thereby providing a model to study early events of neurodegenerative processes. Thus our work opens new perspectives for both fundamental and applied cell targeting protocols using proteic cargoes which need to retain full bioactivity upon internalisation, as illustrated here with the oligomeric Cre recombinase.
Gitton
Yorick
Y
Evolution des Régulations Endocriniennes, CNRS UMR 7221, Muséum National d'Histoire Naturelle, 7 rue Cuvier,75005 Paris, France. gitton@mnhn.fr
Tibaldi
Lorenzo
L
Dupont
Edmond
E
Levi
Giovanni
G
Joliot
Alain
A
D000818:Animals; D002478:Cells, Cultured; D002490:Central Nervous System / Q000196:embryology / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000294:innervation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D019426:Integrases / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D014161:Transduction, Genetic
D011993:Recombinant Fusion Proteins; C045073:Cre recombinase; D019426:Integrases