Abstract
Background and AimGreen fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP.MethodsHepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison.ResultsAll biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14.ConclusionGFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments using GFP and in interpreting their results.
Highlights
The accumulation of green fluorescent protein (GFP) in cells is widely used as a molecular tag that can be readily visualized under ultraviolet light illumination
At 28 days after hepatocyte transplantations, the rat livers showed very few clusters of GFP-positive hepatocytes and they exhibited cytoplasmic degenerative changes with nuclear debris (Fig. 2). These cells were surrounded by a large number of GFP-negative, small, round lymphocyte-like cells that were positive for CD4 or CD8
No GFP-positive hepatocytes and only a few GFPpositive non-parenchymal cells were detected at 42 days after transplantation
Summary
The accumulation of green fluorescent protein (GFP) in cells is widely used as a molecular tag that can be readily visualized under ultraviolet light illumination. Liver harvested from a GFP-Tg Lewis rat survived long term in a wild-type Lewis rat without the use of an immunosuppressant (our unpublished data). These experimental findings imply that GFP is weakly immunogenic, but that organs or cells expressing GFP can survive at sites where there is a weaker immunological reaction. Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models.
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