M T Lin1, F Wang, J Uitto, K Yoon. 1. Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, 233 S 10th Street, Suite 450 BLSB, Philadelphia, PA 19107, USA.
Abstract
BACKGROUND: Tissue-specific promoters are becoming increasingly important in light of their effects on gene expression in gene therapy experiments. The regulation of gene expression may be as important as the delivery of the gene itself. OBJECTIVES: To determine the effects of the involucrin (INV), keratin 14 (K14) and cytomegalovirus (CMV) promoters on the expression of the reporter gene beta-galactosidase. METHODS: In vivo, plasmid DNA was introduced to BALB/c mice by gene gun. Skin biopsies were taken after 24 h for histology and beta-galactosidase staining. In tissue culture cells, plasmid DNA was introduced by transient transfection to cell lines 293 (transformed primary human embryonal kidney cells), NIH 3T3 (immortalized mouse fibroblasts) and human keratinocytes. Reporter gene expression was assayed by histochemical staining and chemiluminescence. RESULTS: The K14 and INV promoter constructs showed beta-galactosidase gene expression only in the epidermis, while the CMV promoter showed gene expression in both the dermis and epidermis. In cell culture, the INV and K14 promoter constructs demonstrated significant beta-galactosidase expression in human keratinocytes, but minimal expression in 293 and NIH 3T3 cell types. CMV promoter constructs demonstrated significant expression in all cell types. CONCLUSIONS: Gene expression can be regulated by different promoters both in vivo and in cell culture. Based on the physiological expression of the different promoters, gene expression can be restricted to certain cell types, tissues and skin layers.
BACKGROUND: Tissue-specific promoters are becoming increasingly important in light of their effects on gene expression in gene therapy experiments. The regulation of gene expression may be as important as the delivery of the gene itself. OBJECTIVES: To determine the effects of the involucrin (INV), keratin 14 (K14) and cytomegalovirus (CMV) promoters on the expression of the reporter gene beta-galactosidase. METHODS: In vivo, plasmid DNA was introduced to BALB/c mice by gene gun. Skin biopsies were taken after 24 h for histology and beta-galactosidase staining. In tissue culture cells, plasmid DNA was introduced by transient transfection to cell lines 293 (transformed primary human embryonal kidney cells), NIH 3T3 (immortalized mouse fibroblasts) and human keratinocytes. Reporter gene expression was assayed by histochemical staining and chemiluminescence. RESULTS: The K14 and INV promoter constructs showed beta-galactosidase gene expression only in the epidermis, while the CMV promoter showed gene expression in both the dermis and epidermis. In cell culture, the INV and K14 promoter constructs demonstrated significant beta-galactosidase expression in human keratinocytes, but minimal expression in 293 and NIH 3T3 cell types. CMV promoter constructs demonstrated significant expression in all cell types. CONCLUSIONS: Gene expression can be regulated by different promoters both in vivo and in cell culture. Based on the physiological expression of the different promoters, gene expression can be restricted to certain cell types, tissues and skin layers.
Authors: John R Zibert; Katrin Wallbrecht; Margarete Schön; Lluis M Mir; Grete K Jacobsen; Veronique Trochon-Joseph; Céline Bouquet; Louise S Villadsen; Ruggero Cadossi; Lone Skov; Michael P Schön Journal: J Clin Invest Date: 2010-12-06 Impact factor: 14.808
Authors: Emilio González-González; Yeu-Chun Kim; Tycho J Speaker; Robyn P Hickerson; Ryan Spitler; James C Birchall; Maria Fernanda Lara; Rong-Hua Hu; Yanhua Liang; Nancy Kirkiles-Smith; Mark R Prausnitz; Leonard M Milstone; Christopher H Contag; Roger L Kaspar Journal: Sci Rep Date: 2011-11-15 Impact factor: 4.379