Green Flourescent Protein Expression in Bone Marrow-Derived Mesenchymal Stem Cells of Immunocompetent and Athymic Nude Rats
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Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Animal Sciences Honors Theses; 2006
Concerns exist regarding potential differences in transgene product expression following gene delivery in immunocompetent versus immunocompromized animal models. To address this question in vitro, this study evaluated expression of green flourescent protein (GFP) in bone marrow-derived mesenchymal stem cells (BMDMSC) from immuncompetent and athymic nude rats following GFP gene delivery using a first-generation adenoviral vector. Cell types used were from 2-4-week old Wistar (Wstr) rats and 10-week-old NIH-rnu nude rats (Nude) of both sexes, and a BMDMSC from Lewis rats (Tul). Intensity and duration of GFP expression were documented every 48 hours in live cells using an in vivo imaging system with a cooled charge-coupled device camera. Intensity was measured in flux (photons/cm2/second/steradian). Expression was compared between cell types in cells in monolayer and three-dimensional alginate culture conditions. Flux values from days 0, 7, 12, and 22 were used for statistical comparisons. There was no significant difference in GFP expression between groups (immunocompetent versus immunocompromized). Rat breed and immune status had no effect on GFP expression (P >0.8, monolayer; P>.60, alginate). Flux values, expressed as a ratio of median expression in AdGFP-transduced cells to background levels, were as follows: Monolayer Day 0 -7792/1; Day 7 – 2634/1; Day 12 – 2592/1; Day 22 – 3819/1; Alginate Day 0 – 2304/1; Day 7- 529/1; Day 12 – 488/1; Day 22 421/1. Background flux was 2.50x106 for cells in monolayer culture and 1.42x106 for cells in alginate constructs. The AdGFP-transduced cells had 1800-fold greater expression, when compared with background values, in monolayer culture, and 525-fold greater expression in alginate constructs. Cells cultured in monolayer showed significantly greater expression compared to those in alginate constructs (P<0.05). These findings will provide valuable information for selection of optimal target cells for gene delivery using Ad vectors, and may alleviate concerns regarding potential differences in transgene expression between and immunocompromized and immunocompetent animal models.
1st Place, Life Science Division, CFAES Undergraduate Research Forum
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