Gene silencing in a human organotypic skin model.
Here we present a simple and highly reproducible method which allows the study of the effects of a single gene knockdown in an organotypic skin model. Human keratinocytes (KC) were transfected with backbone-modified short interfering RNAs (siRNAs) specific for vascular endothelial growth factor (VEGF) and matriptase-1. Twenty-four hours later the transfected cells were seeded onto fibroblast collagen suspensions and allowed to build up a multilayered epidermis by culture at the air/medium interface for 7 days. Protein expression of both targeted genes remained down-regulated by more than 80% up to 8 days after transfection. As expected, VEGF knockdown by siRNA did not alter epidermis formation in our organotypic skin model. By contrast ablation of matriptase-1 led to aberrant KC differentiation and impaired filaggrin processing and resulted in an epidermal phenotype closely resembling that of matriptase-1 deficient mouse skin. Our results suggest that siRNA-mediated gene silencing is highly efficient in an organotypic skin model and readily allows the assessment of the roles of individual genes during terminal KC differentiation.
Mildner
Michael
M
Department of Dermatology, Medical University of Vienna, Vienna, Austria. michael.mildner@meduniwien.ac.at
Ballaun
Claudia
C
Stichenwirth
Martin
M
Bauer
Reinhard
R
Gmeiner
Ramona
R
Buchberger
Maria
M
Mlitz
Veronika
V
Tschachler
Erwin
E
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D005347:Fibroblasts / Q000502:physiology; D000091344:Filaggrin Proteins; D020868:Gene Silencing; D005821:Genetic Techniques; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D009924:Organ Culture Techniques; D034622:RNA Interference; D012697:Serine Endopeptidases / Q000235:genetics; D012867:Skin; D012879:Skin Physiological Phenomena; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D042461:Vascular Endothelial Growth Factor A; D012697:Serine Endopeptidases; C485595:matriptase