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      Clonal analysis of stably transduced human epidermal stem cells in culture.

      Proceedings of the National Academy of Sciences of the United States of America

      3T3 Cells, Animals, Cell Division, Cell Transplantation, Clone Cells, Coculture Techniques, DNA, analysis, DNA, Complementary, Epidermis, Genes, Reporter, Humans, Interleukin-6, biosynthesis, Keratinocytes, cytology, Mice, Mice, Nude, Recombinant Proteins, Repetitive Sequences, Nucleic Acid, Skin, Stem Cells, Transfection, Transplantation, Heterologous, beta-Galactosidase

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          We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.

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