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Toxic Transfections

Are your cells dying or looking bedraggled after transfection? Unintended toxicity can reduce transfection efficiency, confound interpretation of results, and even preclude analyzing transfected cells altogether if little to no cells remain to assay.

 

In this TransMission Transfection 101 we will cover:

  • Reasons that cells die or exhibit abnormal phenotype after transfection
  • Common methods to assess toxicity
  • How to reduce transfection-related toxicity

 

By the end of this TransMission, we hope you will understand the importance of recognizing transfection-mediated cytotoxicity and strategies for minimizing this frustrating phenomenon.

 

Reasons that cells die or exhibit abnormal phenotype after transfection

Cells in culture, like the cells in our body, harness signaling pathways and innate immunity to detect and respond against potential threats. The very act of transfection—delivering foreign nucleic acids to cells—can trigger stress and innate immune responses. See [1] for an introductory review on how nucleic acids are detected and managed by cellular innate immunity. The magnitude and type of immune or stress response to transfection is largely dependent on the cell type and transfection reagent used.

Some cell types are acutely sensitive to xenobiotics and apoptose in the presence of externally introduced nucleic acids, transfection reagents and/or nucleic acid-reagent complexes. On the other hand, some cell types may show little to no outwardly visible signs of toxicity from transfection. In either case, it is important to include negative controls, e.g. ‘Mock’ transfections, to understand the impact of transfection on the experimental system under study. For gene expression studies, researchers can be more confident that the results of their transfection experiment can be attributed to biological pathways of interest (rather than off-target effects) by limiting and accounting for cytotoxicity. For protein production, stable cell line generation and genome editing experiments, reducing cytotoxicity may also improve process efficiency. Contaminants, such as endotoxin, in the nucleic acid preparation used in the transfection can also exacerbate toxicity problems.

Notably, cell death and diminished health may not be ‘off-target’ effects! Researchers should consider if observed cytotoxicity is related to the underlying biology of the delivered nucleic acid. For example, overexpression of certain genes can trigger apoptosis, so transfection of expression plasmids bearing those genes would expectedly lead to increased cell death. Similarly, transfection of siRNA to knockdown essential genes would expectedly impact cell survival. Including a ‘Mock’ transfection with a control nucleic acid or performing a literature review of the genes and pathways involved with the transfected nucleic acid can be helpful to assess whether underlying biology of the transfected nucleic acid is related to any observed cytotoxicity. In some cases, cell morphology changes are ‘normal’ and do not adversely affect experimental objectives. One such case is in transfection to produce lentivirus pseudotyped with vesicular stomatitis virus G (VSVG); overexpression of VSVG protein is known to cause changes in cell morphology, even resulting in cell-cell fusion, but does not adversely affect viral titers.

Though some degree of cytotoxicity is unavoidable owing to native cellular surveillance mechanisms and response to stimuli, it can be significantly minimized in some circumstances. Jump to ‘How to reduce transfection-related toxicity’ below for a full discussion. Importantly, cytotoxicity should be minimized even before starting transfection. Steps to ensure cell health through proper maintenance and culturing techniques is key.

 

Common methods to assess toxicity

Perhaps the easiest method to check for toxicity is to visually assess your cells by comparing the morphology of untransfected ‘Cells Alone’ and ‘Mock’ controls with that of cells transfected with your nucleic acids of interest. Are your normally adherent cells rounded and floating off the plate? Do cell cultures appear clumpy and less dense than their untransfected counterparts? Dyes that enter and stain dead cells due to a loss of cell membrane integrity, e.g. trypan blue and propidium iodide, can assist with quantitation of cell viability. Defects to cell growth can be detected by taking a cell count and comparing counts for transfected cells and controls.

Cell health can also be assayed through coupling to the differential enzymatic activity of live and dead/dying cells. Common methods with established protocols and commercially available kits include the MTT and LDH assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) is a yellow dye that is processed to a violet-blue product in metabolically active cells. LDH (lactate dehydrogenase) is a redox enzyme that ‘leaks out’ of cells that have lost plasma membrane integrity – as with the MTT assay, its activity can be linked to a colorimetric readout.

Changes to gene expression profiles, as in the stress response, usually occur well before morphological symptoms of toxicity are detectable. With reverse-transcription qPCR/dPCR, expression of select genes can be measured with sequence-specific primers. With microarrays, expression of swathes or panels of multiple genes can be measured at once. Free from reliance on gene-specific complementary primer or hybridizing oligo sequences, next-generation sequencing technologies enable quantitation of the entire transcriptome. In 2012, Mirus Bio investigated the impact of transfection on cytotoxicity by performing differential gene expression analysis of 84 genes involved with the stress response after transfection of a non-coding plasmid in HeLa cells [2]. Genes with over two-fold expression differences between cells receiving transfection complexes (reagent with DNA in Opti-MEM®) versus the negative control (DNA in Opti-MEM®) are graphed below with their expression fold change. As shown, up- and down-regulation of genes can even occur through transfection of a non-coding plasmid, and furthermore, the number of affected genes and magnitude of the effect is related to the reagent used in the transfection.

Graph showing upregulation of stress response genes is dependent on the reagent used in transfection.

Mirus TransIT® Transfection Reagents Minimize the Stress Response in Transfected HeLa Cells. Stress-related gene expression changes were determined by RT-qPCR from total RNA samples harvested from HeLa cells that were transfected with TransIT®-LT1, TransIT®-2020 or Lipofectamine® 2000 at 8 and 24 hours. Eighty-four genes were analyzed using the Human Stress Response 96 StellARray™ (Lonza). At both time points, the number and magnitude of stress-related gene expression changes were lower when cells were transfected with TransIT®-LT1 or TransIT®-2020 than when cells were transfected with Lipofectamine® 2000.

Including control conditions, e.g. ‘Cells Alone’ or ‘Mock,’ can be very helpful when assessing toxicity and troubleshooting. Transfection with a ‘Mock’ control that mimics almost exactly transfection with the gene of interest is especially critical for differential gene expression studies.

 

How to reduce transfection-related toxicity

Now that we’ve discussed reasons for cytotoxicity and methods to measure it, let’s cover potential ways to reduce transfection-related toxicity. Below is a list of potential solutions for improving viability and reducing the chance for off-target effects after transfection:

  • If toxicity is new or sudden in an established transfection experiment, carefully consider any changes that have been made in the lab or experiment, e.g. new plasmid preparation or different batch of FBS.
  • Ensure the transfected nucleic acids are free from contaminants such as endotoxin that may carryover from E. coli during the plasmid preparation process. Many commercially available plasmid preparation kits include steps to remove endotoxin.
  • If toxicity is only observed when transfecting a particular cell type or cell line, you may be working with an inherently ‘sensitive’ cell type or using an incompatible transfection reagent. Trying a different reagent or method may be helpful if all other efforts to reduce toxicity are fruitless.
  • Though a culture medium change is not required nor even recommended for transfections with TransIT® reagents, replacing the media post-transfection to remove transfection complexes and replenish nutrients can sometimes improve cell health. If performing a media change, do so any time 4 hours post-transfection to allow uptake of transfection complexes.
  • In some cases, reducing the amount of transfection complex added to cells can greatly reduce cytotoxicity. Experiment with reagent volume and nucleic acid mass to identify ideal transfection conditions. For plasmid transfections, download this protocol (PDF) that outlines an experiment for optimizing reagent volume and plasmid DNA mass used in a transfection.
  • Starting with healthy cells usually helps with toxicity issues. Factors such as cell density or confluency at transfection can impact cell health as well as transfection efficiency. If necessary, thaw a new aliquot of cells if their normal growth behavior has changed.
  • Finally, for adherent cell types, avoid blasting cells off the plate with your pipettor when adding transfection complexes!

Did this post help you with your cytotoxicity problems? Let us know by emailing techsupport@mirusbio.com. We’d love to hear from you.

References

  1. Dempsey and A. G. Bowie, Virology (2015).
    DOI: 10.1016/j.virol.2015.03.013
  2. Khodthong, C., et al.“The Impact of Transfection Mediated Toxicity” (PDF) (2012).
    DOI: N/A

Explore Related Info & Links

  • Download this white paper on ‘The Impact of Transfection Mediated Toxicity’ (2012)
  • Learn about TransIT®-LT1, a transfection reagent so named for its Low Toxicity
  • Check out the Transfection Troubleshooting Checklist here

The TransMission
Feedback or questions? We’d love to hear from you. Email techsupport@mirusbio.com or call us at 888.530.0801.