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Gene & Protein in Disease Transfection methods for TK6 cells

Table 1. The effects of different transfection methods of TK6 human lymphoblasts on transfection efficiency and cell viability
Transfection method Luciferase signal (RLU) Cell viability (%) GFP transfection efficiency (%)
Metafectene Pro - 90 – 90 77 – 83
Lipofectamine LTX 1.6×10 −1.8×10 5 95 – 98 76 – 83
5
Amaxa Nucleofection 2.4×10 −8.2×10 5 80 – 85 70 – 80
5
Abbreviations: RLU: Relative luminescence unit; GFP: Green fluorescent protein.

A B several different cationic categories (Metafectene Pro and
Lipofectamine LTX) at delivering DNA into a TK6 human
lymphoblast.
In our study, Metafectene Pro resulted in highly effective
transfection of plasma DNA with low toxicity into TK6
human lymphoblasts resulting in high percent cell viability
and transfection efficiency. However, no luciferase activity
was detected in TK6 cells transfected with Metafectene.
C The absence of luciferase activity when transfected with
Metafectene Pro suggests that the presence of certain
chemotypes in Metafectene inhibits or interferes with
NanoLuciferase luciferase activity in Nano-Glo Luciferase.
Some inhibitors of NanoLuc include those with a phenyl-
1,4-dihydropyridine found in the drug isradipine and
[53]
aryl sulfonamide . Interestingly, studies showed that
luciferase activities were detected when the same cells were
[54]
cotransfection with GFP-Max . Furthermore, luciferase
Figure 6. Green fluorescent protein fluorescence of different transfection activities have been detected in other systems, such as
methods determined at 24 h. (A) Metafectene Pro, (B) Lipofectatmine the Dual-Luciferase Reporter Assay System, Bright-Glo
®
LTX. (C) Nucleofection. Microscope magnification: ×20. [55,56]
reagent, and Renilla Luciferase Assay System . Due to
high transfection efficiency associated with low toxicity,
methods to avoid the issues associated with viral-based Metafectene Pro has successfully been used to transfect a
transfection methods potentially. wide variety of cell lines [57,58] .
Optimization is typically required to arrive at the best We also found that transfecting at a reagent-to-DNA
transfection conditions for cells. TK6 human lymphoblast ratio of 3:1 was the optimum for our system after 24- and
cell line is a traditionally difficult-to-transfect cell type. 36-h incubation. In contrast, the reagent-to-DNA ratio
Optimizing TK6 human lymphoblasts with nucleic acid of 6:1 was more toxic to our cells. A higher reagent-to-
molecules of interest at a relatively high efficiency while DNA ratio is known to be associated with high toxicity
maintaining cell viability is essential for studying gene in cells . For a transfection reagent to be useful in our
[59]
function, regulation, and protein function. In this study, system, the reagent should exhibit high percentage of
we evaluated optimum conditions for transfection of cell viability, transfection efficiency, and high luciferase
TK6 human lymphoblasts using three commonly used activity. Metafectene did not meet all these conditions
transfection agents: Amaxa Nucleofector Solutions, since no NanoLuciferase activity was detected under
Lipofectamine LTX, and Metafectene Pro. These reagents optimal transfection conditions. Due to the absence of
were selected based on the available information from the detectable luciferase activity associated with Metafectene
respective company concerning their high transfection Pro, we decided to optimize transfection conditions using
efficiency and low toxicity in multiple cell lines, including Lipofectamine LTX and Amaxa Nucleofection Shuttle
difficult-to-transfect cell lines [20,28,49-52] . We assessed System.
the results to confirm that our conditions maximized
both transfection efficiency and cell viability. The data Lipofectamine reagents are associated with relatively
demonstrated that by optimizing transfection conditions high transfection efficiency in many different cell types,
for TK6 human lymphoblasts, nucleic acid molecules can including lymphocytes [60-62] . These reagents are non-
be delivered in a highly efficient manner. Nucleofection is infectious, easy to use, and can transfer DNA of various
more effective than chemical transfection reagents from sizes. High cellular transfection efficiency is attributed to

Volume 2 Issue 2 (2023) 8 https://doi.org/10.36922/gpd.0353