ViaFect™ Reagent: Building Assays in Difficult Cells

Transfection can sometimes seem more like an art than a science—the perfect transfection experiment being dependent on optimization of conditions, including cell density, transfection reagent and DNA:reagent ratio. No one reagent is perfect for every cell type, so there is the added challenge of optimizing performance in your cell line of choice—which may fall into the well-populated “difficult-to-transfect” category  that includes many primary cells.

Among transfection reagents, Lipofectamine® (Thermofisher), and FuGENE® (Promega) are popular and widely used choices. Viafect™ Transfection Reagent is newer and less well-known, but gaining popularity as a high-performance, low-toxicity reagent that performs well across a wide range of cell lines. In head-to-head comparisons with FuGENE and Lipofectamine, Viafect outperformed or equaled the others for expression of transfected reporter genes and resulting cell viability (see the data in this article).

The story of ViaFect begins with Promega Custom Assay Services (CAS), a group that uses Promega technologies to construct made-to-order assays, typically in a cell line. Many projects from the CAS group involve transfecting cells with expression vectors and reporter vectors. In some instances, customers contact CAS to have an assay constructed in a difficult cell line, after attempting and failing, or experiencing difficulty building the assay themselves.

CAS projects start with a proof-of-concept experiment using transient transfection before moving on to production of a clonal, stable cell line. For difficult cell lines, the CAS group previously turned to electroporation after exhausting lipid-based transfection options. Electroporation often worked, but success came with a price—cytotoxicity. The CAS group challenged R&D to find a better solution—better transfection with low toxicity for difficult-to-use cells. The result of that challenge is the ViaFect™ Transfection Reagent.

The figure demonstrates how ViaFect™ Transfection Reagent helped confirmed the concept, with a usable transient transfection prior to the selection of stable TF-1 suspension cells. On the other hand, as shown, electroporation did not result in significant transfection of the cells.

 Figure 1. ViaFect™ Transfection Reagent enables an assay for cytokine signaling in a hematopoietic cell model. TF-1 suspension cells were transiently transfected with pGL4.32[luc2P/NF-κB-RE/Hygro] Vector, an NF-κB response element luciferase reporter, using either ViaFect™ Transfection Reagent at a 2:1 reagent:DNA ratio or Amaxa Nucleofector® II (electroporation). The following day cells were stimulated with TNFα for 6 hours, and the response was measured with Bio-Glo™ Luciferase Reagent.


Figure 1. ViaFect™ Transfection Reagent enables an assay for cytokine signaling in a hematopoietic cell model. TF-1 suspension cells were transiently transfected with pGL4.32[luc2P/NF-κB-RE/Hygro] Vector, an NF-κB response element luciferase reporter, using either ViaFect™ Transfection Reagent at a 2:1 reagent:DNA ratio or Amaxa Nucleofector® II (electroporation). The following day cells were stimulated with TNFα for 6 hours, and the response was measured with Bio-Glo™ Luciferase Reagent.

Primary cells are often difficult to transfect due to their slow growth and high rate of death. ViaFect™ Reagent can be the perfect solution for such cells.  Here’s a selection of recent peer-reviewed articles demonstrating use of ViaFect™ Reagent for transfection of primary cells. For an extensive listing of citations, and to see more data using ViaFect,  check out the product information on the Promega website.

 

Cells Citation
H5 Mouse Chondrocytes Kalev-Zylinska, M.L., et al. (2018) Altered N-methyl D-aspartate receptor subunit expression causes changes to the circadian clock and cell phenotype in osteoarthritic chondrocytes. Osteoarthr. Cartilage https://doi.org/10.1016/j.joca.2018.06.015
iCell Cardiomyocytes Hall, A.R., et al. (2018) Visualizing mutation-specific differences in the trafficking-deficient phenotype of Kv11.1 proteins linked to long QT syndrome type 2. Front. Physiol. 9, 284.
Bovine Aortic Epithelial Cells Gupta, R.M.,. et al. (2017) A genetic variant associated with five vascular diseases is a distal regulator of endothelin-1 gene expression. Cell 170, 522-33.
Human Podocytes Fan, Y., et al. (2017) Rtn1a-mediated endoplasmic reticulum stress in podocyte injury and diabetic nephropathy. Sci. Reports 7, 323.
Murine Neural Stem Cells Bansod, S., Kageyama, R., and Ohtsuka, T. (2017) Hes5 regulates the transition timing of neurogenesis and gliogenesis in mammalian neocortical development. Development 144, 3156-67.
Mouse Cortical Neurons Egusa, S.F. et al. (2016) Classic cadherin expressions balance postnatal neuronal positioning and dendrite dynamics to elaborate the specific cytoarchitecture of the mouse cortical area. Neurosci. Res. 105, 49–64.
Mouse Embryonic Fibroblasts Peng, Y. et al. (2016) AGE-RAGE signal generates a specific NF-κB RelA “barcode” that directs collagen I expression. Sci. Rep. 6, 18822.
Mouse Bone Marrow Macrophages Naujoks, J. et al. (2016) IFNs modify the proteome of Legionella-containing vacuoles and restrict infection via IRG1-derived itaconic acid. PLoS Pathogens 12, e1005408.
Mouse Alveolar Macrophages
Rat Aortic Smooth Muscle Cells Horita, H. et al. (2016) Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation. Nat. Comm. 7, 10830.
Human Renal Proximal Tubule Epithelial Cells Bethge, T. et al. (2015) Sp1 sites in the noncoding control region of BK polyomavirus are key regulators of bidirectional viral early and late gene expression. J. Virol. 89, 3396–411.
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Kyle Hooper

A former Technical Services Scientist, Kyle has also worked with R&D for product development and now specializes in supporting Promega cellular analysis products in North America.

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