Clonal analysis of stably transduced human epidermal stem cells in culture

Monica B. Mathor, Giuliana Ferrari, Elena Dellambra, Michele Cilli, Fulvio Mavilio, Ranieri Cancedda, Michele De Luca

Research output: Contribution to journalArticlepeer-review

Abstract

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial β-galactosidase (β-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 clonogenie keratinocytes for β-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 transgent 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.

Original languageEnglish
Pages (from-to)10371-10376
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number19
DOIs
Publication statusPublished - Sep 17 1996

Keywords

  • epidermis
  • gene therapy
  • keratinocyte

ASJC Scopus subject areas

  • Genetics
  • General

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