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TECHNOLOGY
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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. |
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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.
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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. |
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FIGURE 1
PORE™ RESULTS IN MORE EFFICIENT TRANSFECTION OF MESENCHYMAL STEM CELLS |
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Mesenchymal stem cells
are shown at 24 hours after transfection with green fluorescent
(GFP) gene.
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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 |
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FIGURE 2
TRANSFECTION WITH PORE™ LEADS TO INCREASED LUCIFERASE PRODUCTION IN MESENCHYMAL STEM CELLS |
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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.
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FIGURE 3
PORE™ MAINTAINS BETTER LONG-TERM GROWTH POTENTIAL IN HUMAN MESENCHYMAL STEM CELLS |
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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).
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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.
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FIGURE 4
PORE™ IS EFFECTIVE IN HARD TO TRANSFECT CELL LINES |
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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.
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FIGURE 5
PORE™ ALLOWS SCALABILITY IN TRANSFECTION |
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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.
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FIGURE 6
PORE™ PROMOTES TRANSFECTION OF COMPLEX GENE POOLS |
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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.
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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.
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