The majority of keratinocytes incorporate intradermally injected plasmid DNA regardless of size but only a small proportion of cells can express the gene product.
The expression of intradermally injected DNA by keratinocytes is found mainly in the upper and middle layers of the epidermis. To investigate the mechanism of this selective expression, we observed the sequential changes in the distribution of interleukin-6-expressing keratinocytes after the introduction of the interleukin-6 gene. Transgene expression first occurred in basal keratinocytes and subsequently expanded to all epidermal layers and then remained in the upper layers. Semiquantitative analysis indicated that keratinocytes in the lower layers incorporated and lost DNA earlier than those in the upper layers. In order to examine the effect of the DNA size on the transgene expression, we constructed a plasmid containing a full-length 9 kb cDNA of type VII collagen and introduced it into keratinocytes. The expression pattern of type VII collagen in the epidermis was the same as those for smaller genes. This suggests that plasmid size has little or no effect on the expression pattern of the transfected gene. To trace the introduced plasmid, we intradermally injected a green fluorescence protein expression plasmid coupled with a rhodamine flag. Almost all keratinocytes in the injected areas showed rhodamine fluorescence. Furthermore, some cells also expressed green fluorescence protein. A lack of rhodamine fluorescence in the nucleus suggested an impairment of plasmid DNA transport from the cytoplasm to the nucleus. Collectively, our results show that the majority of keratinocytes take up the intradermally injected DNA regardless of its size, but that the transfer of DNA from the cytoplasm to the nucleus is limiting the transgene expression.
Sawamura
Daisuke
D
Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan. smartdai@med.hokidai.ac.jp
Yasukawa
Kana
K
Kodama
Kazuo
K
Yokota
Koichi
K
Sato-Matsumura
Kazuko C
KC
Toshihiro
Tanaka
T
Shimizu
Hiroshi
H
D000818:Animals; D024143:Collagen Type VII / Q000235:genetics; D000078404:Epidermal Cells; D015870:Gene Expression / Q000502:physiology; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007271:Injections, Intradermal; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000493:pharmacokinetics*; D051381:Rats; D011919:Rats, Inbred Strains; D019076:Transgenes / Q000235:genetics
D024143:Collagen Type VII