The ease with which cells of the skin can be grown in tissue culture and genetically modified using retroviral-method gene transfer have raised the possibility of using modified skin grafts as a means to correct genetic disease where the skin is the major afflicted tissue or where modified skin grafts are used to synthesized and deliver a missing plasma protein, whatever the application, a key to the success of these grafts is the genetic modification of those skin cells which will persist long term after transplantation and express the new gene faithfully. For the epidermis, the hypothesis is that epidermal stem cells can be propagated in vitro, genetically modified and transplanted to generate a long lasting self-renewable tissue capable of correcting disease. This application proposes to test this hypothesis by using retroviral- mediated gene transfer as a unique tool to gene mark skin cells in order to delineate the fate and growth potential of these cells in vitro and in vivo. Since recombinant retroviruses integrate at random sites in the genome of target cells it's possible not only to permanently mark entire population of cells but also to identify specific clones of cells and their respective progeny each clone's distinctive site of integrated virus. In this way stem cells, their tissue organization in the epidermis and the factors that influence their proliferation in tissue culture and post-transplantation can be identified. As a corollary to this project, this application proposes to monitor the stability of expression of integrated recombinant viruses in cells during extended in vitro cultivation and during long term function as modified skin grafts. Taken together, these tow components should improve our understanding of the unique physiology of stem cells of the skin as well as the ability of skin cells to express new genetic information. It is believed that these are two important components of the success of a genetically modified skin graft for the correction of disease.
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