title: Nucleofection: a new, highly efficient transfection method for primary human keratinocytes*.
abstract: Transfection is an essential tool for numerous in vitro applications including studies of gene expression, promoter analysis, and intracellular signaling pathways and also for therapeutic strategies such as tissue engineering and gene therapy. However, transfection of primary cells including keratinocytes with common methods such as calcium phosphate, DEAE-dextran, liposome-mediated transfer, electroporation or viral vectors is problematic because of low transfection efficiency and the induction of terminal differentiation. Here we analyzed the use of nucleofection, a new, electroporation-based transfection method that enables the DNA to enter directly the nucleus, for the transfection of keratinocytes. Several different conditions were tested and optimized, resulting in a final transfection efficiency of 56% in primary human epidermal keratinocytes. This efficiency is superior to all non-viral transfection methods reported so far. The number of non-viable keratinocytes after nucleofection was low, varying between 14 and 16%. In contrast to other transfection protocols, nucleofection did not induce terminal differentiation in the transfected keratinocytes. In addition, nucleofection is a fast method, because the results can be analyzed within 7 h. In summary, nucleofection is a fast, easy and highly effective alternative for the transfection of primary human keratinocytes, which offers new opportunities for various research applications.
authors.affiliation: Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland.
authors.forename: Jörg H W
authors.initials: JH
authors.lastname: Distler
authors.forename: Astrid
authors.initials: A
authors.lastname: Jüngel
authors.forename: Mariola
authors.initials: M
authors.lastname: Kurowska-Stolarska
authors.forename: Beat A
authors.initials: BA
authors.lastname: Michel
authors.forename: Renate E
authors.initials: RE
authors.lastname: Gay
authors.forename: Steffen
authors.initials: S
authors.lastname: Gay
authors.forename: Oliver
authors.initials: O
authors.lastname: Distler
mesh_terms: D052160:Benzothiazoles; D015153:Blotting, Western; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism*; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology; D003637:DEAE-Dextran / Q000737:chemistry; D003959:Diamines; D018274:Electroporation; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D008081:Liposomes / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009930:Organic Chemicals / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011804:Quinolines; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D004396:Coloring Agents; D003959:Diamines; D008081:Liposomes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D003637:DEAE-Dextran