Establishment of effective gene transfer method to three-dimensional skin culture

Gene engineering techniques such as transgenic and gene knock-out animals are powerful tools to clarify gene function in the organism. It is known that a conserved gene between animals and humans plays a physiologically distinct role in the growth and differentiation of cells. Elucidation of the gene function in the human body is valuable for the development of novel medicines such as molecular target drugs. However, it is extremely difficult to engineer certain genes in humans. We present here an effective gene transfer method to three-dimensional skin culture (3D culture) with replicativedeficient adenovirus vector. The 3D culture is a human skin equivalent consisting of stratified keratinocytes and a dermal equivalent. We added the adenovirus vector onto the keratinocytes and into the cultured medium before starting the stratification. The expression level of the introduced gene continued at least 10 days after infection. Furthermore, we investigated the role of protein kinase C (PKC) in the 3D culture with this method. We introduced the adenovirus expressing the dominant negative mutants (D/ N) of PKC α, δ, ε and η to the 3D culture and examined their effects on growth and differentiation. D/ N PKC α increased proliferation of the basal cells of keratinocytes, and D/N PKC δ caused irregular growth of some cells in the spinous layer. D/N PKCδ increased loricrin expression, a late marker of keratinocyte differentiation, whereas D/N PKCη decreased the loricrin expression. These findings indicate that a combination of 3D culture with the adenovirus vector is a useful tool for elucidating the gene function.