Page 58 - GPD-2-2

Gene & Protein in Disease Transfection methods for TK6 cells

reagent/DNA ratio of 3:1 when 1.0 µg of DNA is utilized A
for transfection; no significant difference in transfection
efficiency and cell viability was observed between 24 and
36 h transfection times.
The experiment performed in Figure 1 was repeated by
utilizing the best transfection conditions (reagent to DNA
ratio of 3, 1 µg of DNA) obtained for the Metafectene Pro
reagent. Cell viability and transfection efficiency were
then assessed using GFP and NanoLuciferase activity
(Figure 2). Cells transfected with pNL1.1CMV had
no significant effect on cell viability (Figure 2A). The
transfection efficiency of cells that received the pGFPmax
plasmid was significantly greater than control cells that
received no DNA (p < 0.05, Figure 2B). No luciferase B
activity was detected (Figure 2C). Collectively, these
results demonstrate that Metafectene Pro is not toxic
to cells under conditions where 80% GFP transfection
efficiency is achieved. Since no NanoLuciferase activity
was detected under optimal transfection conditions
for this reagent, Metafectene Pro cannot be used in
our system. For a transfection reagent to be useful
in our system, the reagent should be able to produce
high transfection efficiency and luciferase activity with
minimal toxicity.
3.2. Optimizing transfection conditions for
lipofectamine LTX
To determine the toxicity and transfection efficiency of C
lipofectamine LTX, cells were seeded in 6-well plates at
0.5 × 10 and 1.0 × 10 cells/well and transfected with 0.5
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µg and 1.0 µg of pGFPmax plasmids, respectively. Cells
transfected with 1.0 µg of plasmid showed no significant
difference in cell viability between the control cells that
received no DNA and cells transfected with DNA at
24 and 36 h post-exposure (p > 0.05, Figure 3A). Cells
transfected with pGFPmax demonstrated a significantly
greater transfection efficiency than the control cells
(p < 0.05, Figure 3B). In a parallel experiment, the cells
were transfected with 1.0 µg of pNL1.1CMV plasmid,
while control cells received 1.0 µg promoterless vector
pNL1.2. No significant difference in cell viability between
cells transfected with pNL1.1CMV and control cells Figure 2. Transfection efficiency of Metafectene Pro. TK6 human
lymphoblast cells were subjected to transfection using Metafectene Pro .
®
that received pNL1.2 at 24 and 36 h post-transfection (A) Cell viability. (B) Green fluorescent protein transfection efficiency.
(Figure 3C). Luciferase activity was significantly greater in (C) Luciferase activity. *p < 0.05.
cells transfected with pNL1.1CMV than the control cells
at 24 and 36 h post-transfection (p < 0.05, Figure 3D); a conditions, cells express NanoLuciferase to the order of
significant 30% increase in luciferase activity was observed 1.8 × 10 RLU, indicating that lipofectamine LTX could
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at 36 h as compared to 24 h post-transfection (Figure 3D). potentially be utilized for our NanoLuciferase promoter
Collectively, these results demonstrate that lipofectamine assays, especially at 36 h post-transfections, when the
LTX achieves transfection efficiency of at least 80% with promoter activity is at its highest levels; 1.0 × 10 cells/well
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no significant toxicity to TK6 cells at both 24 and 36 h transfected with 1.0 µg plasmid DNA seems to achieve the
transfection times. Under lipofectamine transfection highest number of transfected cells.

Volume 2 Issue 2 (2023) 5 https://doi.org/10.36922/gpd.0353