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From: Kuch
Date: 5/14/2002
Time: 1:42:27 PM
Remote Name: 66.88.192.59
Abstract TITLE: Survival of genetically-modified fibroblasts in vivo as a function of cloning, transduction and cellular lifespan. AUTHORS (ALL): Tandeski, Terry 1; Jorgensen, Cynthia 1; Chen, Mei 2; Morgan, Jeffrey 3; Petersen, Marta 1; Krueger, Gerald 1 INSTITUTIONS (ALL): 1. Dermatology, University of Utah, Salt Lake City, UT, USA; 2. Dermatology, University of Southern California, Pasadena, CA, USA; 3. Research Unit, Shriners Burn Institute, Boston, MA, USA;
ABSTRACT BODY: Abstract Body: We have shown previously that genetically modified human fibroblasts (GMFb) seeded into a biocompatible nylon mesh (skin construct) lose transgene expression and are replaced by host fibroblasts when transplanted in vivo. In these studies, GMFb were cloned by limiting dilution before seeding the matrix. We hypothesize that loss of transgene expression and survival (TGE/S) in vivo may result from the process of cloning or transduction. To test this hypothesis, cloned and/or transduced fibroblasts from the same source were used to seed nylon mesh matrices. Fibroblasts studied included normal human fibroblasts (Fb) passaged 17 times, cloned Fb passaged 17 times, cloned lacZ-transduced Fb passaged 17 times, cloned Fb transduced with a retroviral vector encoding human telomerase (hTRT) passaged 17 or 70 times, and cloned Fb transduced with hTRT and lacZ passaged 70 times. Skin constructs were matured in vitro for 1 month, transplanted to nude mice and harvested after 4 and 8 weeks in vivo. After harvesting, Fb survival was assessed by quantifying the number of murine (host) vs human cells in the skin constructs. These analyses demonstrated: a) there is no difference in survival between non-cloned and cloned Fb; b) there is no difference in survival between lacZ-transduced Fb compared with non-transduced or non-cloned Fb; c) Fb transduced with hTRT survived considerably longer in vivo than those not expressing hTRT. The survival advantage of hTRT-transduced Fb was most pronounced in cells passaged 70 times. Differences were seen after at 4 weeks in vivo. We conclude that transduction and/or cloning of Fb does not adversely affect cell survival in vivo, and that the inherent lifespan of the cell is most critical to cell survival in this system.
Kuch
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Time: 21:26:24
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