Transfection Efficiencies

Transfection Efficiency is an important metric in cellular genetic engineering and finds applications in the development of biologics (including vaccine and antibody production), CAR T-cell therapies and other cell therapies.  Researchers strive for high transfection efficiencies and high viability but this is a laborious process. With traditional techniques, cells are plated immediately post-electroporation. 1-2 days later, cells are stained to determine if the receptor or antibody of interest is being expressed. This is a time-consuming process and can be very inefficient if poor transfection efficiencies are achieved.

 

Inish Solutions for Transfection Efficiency Prediction (TEP)

Cellix's Inish Analyser predicts transfection efficiencies immediately post-transfection by giving an accurate analysis of the percentage of cells with open-membranes. We call this the Transfection Efficiency Prediction (TEP) assay. The TEP assay is run immediately post electroporation enabling you to determine transfection efficiencies without having to wait for receptor/antibody expression. This saves time and improves workflows ensuring you are plating the highest number of transfected cells.  

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Results

Results are displayed in the form of a scatterplot and in a table.

Transfection Efficiency of Jurkat T-cells (high viability)
Transfection Efficiency of Jurkat T-cells (high viability)

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Transfection Efficiency of Jurkat T-cells (high expression)
Transfection Efficiency of Jurkat T-cells (high expression)

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Transfection Efficiency of Jurkat T-cells (high viability)
Transfection Efficiency of Jurkat T-cells (high viability)

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On the scatterplot, there may be up to 4 visible populations depending on your restuls:

  • “Live – Membrane closed” cell population is represented by the population of green dots.  Although these are live cells, they are unsuccessfully transfected cells as the membrane failed to open in order to receive the vector/DNA.​

  • “Live - Membrane open” cell population is represented by the population of blue dots.  This is your population of successfully transfected cells.  As the cell membrane has been successfully opened, these cells can uptake the vector.  The ability to analyse and sort cells of this type is totally unique to Cellix!

  • Dead cell population is represented by the red dots.

  • Debris population is represented by the population of dots on the left-hand side of the scatterplot along the Y-axis.

Transfection Efficiency_Results_Populations labelled.jpg

Gating Populations

"Gating” the populations means selecting the population(s) of interest for analysis. For the TEP Assay, this means selecting the “Live - Membrane open” cell population.

  • Top Green Horizontal Line: Above the line is where the Live cell population is positioned and below the line is where the Live - Membrane open cell population is positioned.

  • Bottom Red Horizontal Line: Above this is where the Live - Membrane open cell population is positioned and below the line is where the Dead cell population is positioned.

  • Vertical Line: Left-hand side of this line is where the Debris population is positioned.

 

Horizontal and vertical lines may be moved by placing your finger on the line and sliding it up/down or left/right on the scatterplot. The accompanying table with all the results will be updated accordingly.

 

Compare with Current Methods

What is Transfection?

Transfection is the process by which the genetic material is delivered into the cell.  Broadly speaking, there are two main methods:  Transfection (non-viral) vs. Transduction (viral).  Up to now, the most common method of gene editing was via the use of viral vectors (i.e. using viruses to deliver the DNA inside the cell), otherwise known as transduction.  Viral vectors run the risk of causing severe immune responses in patients and a number of deaths have been attributed to this in several clinical trials in the past.  However, viral vectors also pose significant manufacturing challenges, as in addition to the cells as starting material, the production process has to take into account the raw materials of viral vectors which are highly complex.  Many industry players now recognise that long-term, successful non-viral transfection methods will be less challenging for manufacturing. 

Problems with determining Transfection Efficiencies

Following electroporation, cells are typically plated in 96-well plates.  1-2 days later, successfully transfected cells begin to express the receptors of interest.  These cell receptors are labelled with a fluorescent dye/stain or magnetic bead which enables fluorescent Flow Cytometers to analyse and quantify the succesfully transfected (i.e. genetically engineered) cells.  

Thus, the move to non-viral transfection methods, in particular electroporation (where an electrical field is applied to cells increasing the permeability of the cell membrane, allowing DNA to be introduced into the cell), is gathering steam in terms of manufacturing capability:  increased reproducibility and quality control.  Electroporation has become increasingly popular in recent times as it is more effective than viral transduction when transfecting larger gene edits, which is also more common when using CRISPR techniques.  One of the most popular electrporators on the market is the Amaxa Nucleofector from Lonza.  However, there are still challenges with electroporation, in particular, primary cells such as T-cells, B-cells or stem cells are notoriously hard to transfect.

Transfection
 

Why Measure Transfection Efficiencies?

Gene Therapy - the reason why transfection effiencies are important

Gene therapy is being heralded as the most disruptive advancement to medicine in our entire history which could result in the eradication of infectious diseases such as malaria, Yellow Fever, Dengue virus or Zika; the elimination of genetically inherited conditions such as cystic fibrosis or Epidermolysis bullosa (“butterfly children”) and amazingly…. curing cancer.

Gene therapy is a technique for correcting defective genes that are responsible for disease development.  Approaches include inserting a normal gene to compensate for a non-functional gene or trading a normal gene for an abnormal gene.  Gene therapy should not be confused with genetically modified organisms (GMOs) which contain ‘foreign DNA’ or ‘transgenes’ from another species.  Gene therapy will change the established healthcare market in profound ways, as many current treatments are replaced by more effective genetic treatments and even cures.  

Gene Therapy

However, while clinical trials show exciting promise, there are significant challenges to be overcome; the (cell) manufacturing of these gene therapies on a production scale is no easy task and differs significantly from traditional drug manufacturing where thousands of batches of one drug, manufactured in well-established and controlled processes, result in millions of doses to treat millions of patients.

Trying to improve your transfection efficiency? So are we. 

All researchers working with cell transfection methods, whether in academia or in industry, know about the pains involved in optimising cell transfection. Sometimes some cells just seem to be more resistant to transfection than others, often for frustratingly vague reasons.  Is poor transfection efficiency wasting your time?  We can help.

Inish Solutions:

1. Increase overall transfection efficiency

2. Increase quality of gene transfection data

3. Fast-track successful gene transfection methods and quickly optimise experimental conditions

 

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