Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system.
Efficient gene delivery to the skin is important for gene therapy of skin diseases and in-depth biologic studies of epidermis. In this report, we investigated three nonviral transfection systems for gene transfer in cultured human keratinocytes and organotypic cultures. SuperFect is a highly branched polycationic transfection reagent, PrimeFector a polycationic liposome compound, and the AVET (adenovirus-enhanced transferrin-mediated) system consists of a ternary complex of biotinylated chemically inactivated adenovirus noncovalently complexed with plasmid DNA and polylysine-transferrin. After AVET transfection of cultured keratinocytes with pCIbetagal, a CMV/beta-galactosidase reporter plasmid, 28.8% +/- 1.4% of the cells were stained blue. SuperFect was about 2-fold less efficient, whereas Primefector did not transfect keratinocytes. Similar results were obtained when transfection efficiencies were measured by enzyme assays. Addition of holotransferrin to the culture medium or replacement of polylysine-transferrin by polylysine in the ternary complex did not affect the transfection efficiency. Using AVET complexes without adenovirus, however, strongly diminished gene delivery. This indicates that the AVET complex is taken up by an adenovirus receptor. Separation of AVET/pCIbetagal transfected keratinocytes by adhesion to collagen IV into two fractions (rapidly and slowly adhering cells) showed that the latter were transfected at a 3-fold higher level. Therefore, it seems that putative stem cells adhering rapidly to collagen IV are not efficiently transfected by AVET. AVET-transfected keratinocytes derived from keratinocyte trans- glutaminase negative lamellar ichthyosis patients with a CMV-TGK expression plasmid showed that it is possible to reach a level of total enzyme activity similar to that found in cultured keratinocytes from normal individuals. In organotypic cultures from outer root sheath cells AVET transfection was not successful, which might be due to the presence of the cornified layer or inaccessibility of the adenovirus receptor. In summary, the AVET system provides a powerful tool for transient in vitro transfection of keratinocytes.
Huber
M
M
Department of Dermatology, DHURDV, Lausanne, Switzerland.
Limat
A
A
Wagner
E
E
Hohl
D
D
D016475:3T3 Cells; D000256:Adenoviridae / Q000502:physiology*; D000818:Animals; D001706:Biopsy; D002478:Cells, Cultured; D006651:Histocytochemistry; D006801:Humans; D017490:Ichthyosis, Lamellar / Q000473:pathology; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D011991:Receptors, Virus / Q000502:physiology*; D014162:Transfection / Q000187:drug effects*; D014168:Transferrin / Q000494:pharmacology
D011991:Receptors, Virus; D014168:Transferrin