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TECHNOLOGY
 
 

PORE™ is a non-viral, electroporation technology that provides the simplest, fastest, and most efficient method of DNA transfer in the market today. Proteacel has validated its PORE™ technology in a variety of cells, including mesenchymal stem cells (MSC), human dendritic cells (DC), human embryonic cells (ES), murine embryonic stem cells (ESC) and the MDA-MB-231 adenocarcinoma cell line. PORE™ boasts higher gene transfer efficiencies and better cell survival rates, along with better long-term growth potential and higher expression levels than the leading competitors for non-viral and viral transfection.

 
 
     
 
In contrast with viral gene transfer technologies, PORE™ produces no permanent genetic modification to transfected cells, poses no known human health risks and operates at low production costs.
 
   
     
 
A comparison with its main competitor for non-viral transfection demonstrates that PORE™ routinely yields higher cell viability, higher numbers of transfected cells, a greater quantity of protein produced from the transfected gene (shown by luciferase production), and better long term growth potential.

Figures 1, 2 and 3 show that Mesenchymal Stem Cells (MSC) transfected with PORE™ have greater survival, better long term growth potential and higher expression levels, respectively, than those transfected with the leading electroporation competitor.

 
 
FIGURE 1
PORE™ RESULTS IN MORE EFFICIENT TRANSFECTION OF MESENCHYMAL STEM CELLS

 
 
 
 
Mesenchymal stem cells are shown at 24 hours after transfection with green fluorescent (GFP) gene.
 
 
A. Untreated control cells
B. Cells transfected using PORE™ shown in phase contrast microscopy
C. Cells transfected using PORE™ shown in fluorescence microscopy
D. Cells transfected with the leading competitor’s electroporation technology in phase      contrast microscopy
E. Cells transfected with the leading competitor’s electroporation technology in      fluorescence microscopy
 
     
 
FIGURE 2
TRANSFECTION WITH PORE™ LEADS TO INCREASED LUCIFERASE PRODUCTION IN MESENCHYMAL STEM CELLS
 
 
 
 
The luciferase gene was transfected into mesenchymal stem cells using PORE™ and the leading competitor’s electroporation technology. Luciferase activity was measured 24 hours after transfection.
 
     
 
FIGURE 3
PORE™ MAINTAINS BETTER LONG-TERM GROWTH POTENTIAL IN HUMAN MESENCHYMAL STEM CELLS
 
   
 
Colony forming potential is lost in mesenchymal stem cells electroporated in competitor’s buffer, but is retained in cells transfected using PORE™ technology. Following transfection, cells were plated in 35mm dishes and grown for 12 days to allow formation of colonies, which were stained with crystal violet (colonies are dark spots and consist of 6-20 cells).
 
     
 
A major advantage of the PORE™ technology is its ability to optimally transfect cell lines that are notoriously resistant to the uptake of DNA, such as human mesenchymal stem cells (MSC) as shown in Figures 1 - 3. Excellent results are also obtained with other difficult to transfect cells (Figure 4) including human dendritic cells (DC), human embryonic cells (ES), and murine embryonic cells (ESC). In addition, researchers pursuing high content screening can reliably depend on PORE™ to permit scalability and complex transfections that together allow for the analyses of several different indicator systems in the same cell.
 
     
 
FIGURE 4
PORE™ IS EFFECTIVE IN HARD TO TRANSFECT CELL LINES
 
 
 
 
Cultures of other difficult to transfect cells including human embryonic stem cells (ESC), human dendritic cells (DC), and murine embryonic stem cells are shown in phase contrast and microscopy 24 hours after GFP transfection using PORE™ technology.
 
     
 
FIGURE 5
PORE™ ALLOWS SCALABILITY IN TRANSFECTION
 
   
 
HEK293 cells were transfected with increasing amounts of plasmid DNA encoding firefly luciferase using PORE™. Luciferase activity was measured 24 hours after transfection. The measured values were plotted against the amount of DNA used in the transfection, revealing a linear increase in enzyme production with increasing amounts of plasmid DNA.
 
     
 
FIGURE 6
PORE™ PROMOTES TRANSFECTION OF COMPLEX GENE POOLS
 
 
 
 
Three pools of five genes encoding FLAG tagged proteins (Groups 1, 2, 3) were formulated to provide a test of multiple gene delivery. A plasmid encoding GFP was included in each pool for tracking. Equal amounts of these three pools were combined and used to create a fourth pool (All) containing 15 genes. A fifth pool, made to contain the same 15 genes also contained a 16th gene encoding a red fluorescent protein (All + RFP). U2OS, an osteosarcoma cell line, was transfected with each pool using PORE™ and allowed to grow for 24 hours before harvesting. The western blot detects the expression of the FLAG-tagged proteins in each pool. Each of the bands apparent in lanes 1-3 (Groups 1, 2, 3) are expressed at similar levels in lane 4 (All) and lane 5 (All+RFP), demonstrating that even large numbers (~16) genes can be delivered using PORE™. To demonstrate that the expression of 16 proteins occurred in each cell, we compared the expression of green fluorescent protein (GFP) and red fluorescent protein (RFP) in cells transfected with the fifth pool (All + RFP). It is apparent that GFP expressing cells also express RFP, suggesting that each cell co-expresses all 16 genes.
 
     
 
Figures 1–6 illustrate a rigorous validation of the PORE™ technology, and demonstrate its superiority over transfection methods that exist in the market today. The benefits of PORE™ are numerous: increased efficiency and reduced toxicity, even in hard to transfect cells; better long-term growth potential; scalability; and co-expression of complex gene pools. In combination, these benefits provide a powerful and long awaited platform for an anxious scientific community to advance research on cell models that are destined to play a key role in drug discovery, toxicology, cell differentiation and cell based therapies.
 
     
 
Please take the time to peruse our Products page which lists Proteacel’s available transfection services and product development opportunities.