Page 55 - GPD-2-2

Gene & Protein in Disease Transfection methods for TK6 cells

vectors are associated with high gene transfer efficiencies. Optimal transfection conditions are those that
However, viral-mediated transfections are labor-intensive yield maximal reporter gene expression with minimal
and require certain biosafety measures. In addition, viral detrimental impact on cell viability. No single delivery
vectors generally transduce reticuloendothelial organs, method or transfection reagent can be applied to all types
which dramatically decreases the delivery efficiency of of cells; cellular cytotoxicity and transfection efficiency vary
viruses into their target organs [13,14] . Non-viral transfection dramatically depending on the reagent, protocol, and cell
[44]
methods are relatively safer but have several drawbacks, type being utilized . The cell line used in this study is TK6
which include inefficiency and toxicity . Non-viral human lymphoblasts, which, like Jurkat cells, is traditionally
[15]
transfection can be explored using physical and chemical difficult to transfect due to its fragility and slow-dividing
approaches. The physical transfection approach uses rates. Transfection of TK6 human lymphoblasts is an
a wide range of physical tools (e.g., needle injection, essential tool for scientific and therapeutical applications.
electroporation, gene gun, ultrasound, and laser-based Herein, we examine the transfection efficiency of three
transfection) to deliver nucleic acid into cells . In a commercially available transfection reagents, Metafectene
[16]
®
™
chemical approach, natural and synthetic chemicals, such Pro , Lipofectamine LTX, and Amaxa Nucleofector
as diethylaminoethyl-dextran, cationic lipids, and cationic Shuttle System, using different buffers. These transfections
polymers, are used to facilitate the delivery of nucleic acid reagents were selected due to their high transfection
into the cell membrane [17,18] . efficiency and minimal cell toxicity [25-34] . Results from this
study will lead to the development of optimized protocols
®
Metafectene Pro is a polycationic transfection reagent for transfection efficiency of hard-to-transfect-cell-line
®
based on liposome technology . Metafectene Pro ensures such as TK6 human lymphoblasts.
[19]
easy entry of plasmid DNA into cells by condensing DNA
into compact structures [19,20] . Metafectene Pro exhibits 2. Materials and methods
®
high transfection efficiency and low toxicity in multiple
cell lines and primary cells, including human embryonic 2.1. Nucleofection systems and reagents
kidney 293 cells (HEK 293), human leukemia monocytic The Amaxa nucleofection solution SF, SE, and SG
cell line (THP-1), immortalized murine microglial cells was obtained from Lonza (Allendale, New Jersey).
derived from C57/BL6 (BV2), primary T-cells, and Jurkat Lipofectamine LTX and Metafectene Pro were purchased
cells [20,21-25] . from Life Technologies and Biontex Inc., respectively.
Lipofectamine LTX with PLUS reagent is an origin- 2.2. Plasmid DNAs
™
™
free liposomal transfection reagent. Lipofectamine
is effective, easy to use, and relatively less expensive The pGFPmax was obtained from Lonza (Allendale, New
compared to the other transfection methods. Furthermore, Jersey). The pGFPmax contains the lac promoter and drives
the expression of an enhanced green fluorescent protein
lipofectamine consistently produces high transfection (GFP). NanoLuciferase reporter vectors pNL1.2 (NlucP)
efficiency [26,27] . The popularity of the use of a liposome- and pNL1.1 (Nluc) under the control of Cytomegalovirus
based transfection, Lipofectamine LTX with PLUS , is promoter (pNL1.1.CMV) were purchased from Promega
™
™
™
based on the number of scientists using this technique for a Corporation (Madison, Wisconsin). The pNL1.1.CMV
variety of cell lines such as human mesenchymal stem cells, contains the CMV promoter and expresses NanoLuciferase,
Jurkat cells, transformed HEK293T cells and Michigan and pNL1.2 (NlucP) is a luminescent reporter.
Cancer Foundation-7 (MCF-7) cells [28-32] .
™
The Amaxa Nucleofector Shuttle System is an 2.3. Cell culture
electroporation technique that utilizes a combination Human p53-proficient B-lymphoblastic TK6 cells were
of electrical parameters generated by a device called generously provided by Dr. Howard Liber, Colorado State
Nucleofector with cell-type specific reagents . University. Cells were passaged at 2.0 ×10 cells/mL in
[33]
5
Nucleofection is famously known to overcome the RPMI 1640 supplemented with 2 mM L-glutamine and
lower transfection efficiency by chemical methods. The 10% fetal bovine serum (FBS, Life Technologies, Inc.).
nucleofection system is a significant advance over standard Cells were incubated at 37°C with 5% CO , and the media
2
electroporation systems for its high transfection efficiency was changed every 36 h. Cells were passage into fresh
(optimized nucleofection parameters yielded survival rates media 12 – 14 h before each experiment.
above 60%) in a multitude of cell lines such as primary
neurons, dendritic cells, T-cells, leukemia cells, peripheral 2.4. Metafectene transfection
blood mononuclear cells, ovarian cancer cell lines, human Optimization was done by following the optimized
myeloma cell lines, and Eimeria [34-43] . protocols for the Jurkat cell line. The TK6 cells were

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