Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 348, Issue 1, Pages 76-82Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2006.07.035
Keywords
RNAi; siRNA; keratinocytes; organotypic skin culture; skin; matriptase-1; VEGF; knockdown
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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. (c) 2006 Elsevier Inc. All rights reserved.
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