Abstract
Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression.
Highlights
Nucleofection is a powerful electroporation-based technique to transfer nucleic acids (DNA, RNA) into cells (Trompeter, Weinhold, Thiel, Wernet, & Uhrberg, 2003)
The goal of this study was to determine, if the primary cells derived from sheep skin stored in refrigerator for different time intervals can be transfected effectively by Amaxa nucleofection system
Using the standard protocol and COS 7 program, we observed that the green fluorescent protein (GFP) gene containing plasmid DNA when transferred into primary fibroblast cells using a nucleofection technology expressed in all 3 sheep cell-lines tested, irrespective of their postmortem time interval between animal slaughter and cell culture from refrigerated explants (Table 1, Figure 1)
Summary
Nucleofection is a powerful electroporation-based technique to transfer nucleic acids (DNA, RNA) into cells (Trompeter, Weinhold, Thiel, Wernet, & Uhrberg, 2003). Fibroblasts are a powerful tool for the study of ex-vivo delivery of nucleic acids, including genes for therapeutic proteins, to design cellular therapies and tissue engineering products (Dickens et al, 2010; Koster & Waterham, 2017; Zhang et al, 2011). Their transfection efficiencies, using non-viral methods, are low and vary for different cell types and species, necessitating their prior optimization for the desired species/cell type. We show efficient transfection of sheep dermal primary fibroblasts using nucleofector technology
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