September 1, 2010 (Vol. 30, No. 15)

James Netterwald, Ph.D.

Improved and New Reagents Facilitate Work with Difficult-to-Transfect Cells

Ever since the discovery of DNA’s double-helix structure, life scientists have been interested in somehow introducing foreign nucleic acid molecules into a host cell and expressing the genes encoded by it. Transfection is one of the most popular methods for doing so in mammalian cells.

There are currently several transfection reagents available for research use, and more were recently introduced at the Society for Biomolecular Sciences (SBS) annual meeting. The meeting provided an update on transfection technology and reviewed some of the most recent additions including both electroporation-based and chemical-based transfection technologies.

At the SBS meeting, James Brady, Ph.D., director of technical applications at MaxCyte, presented data on a rapid, automated, and scalable electroporation-based transfection technology. It was originally developed for clinical applications but now has broader research applications including high-throughput drug screening.

The basic experimental outline for using this technology for clinical applications is: select patients with a specific disease; remove cells from the patient; transfect with plasmid DNA, messenger RNA, siRNA, proteins, or small molecules that enhance the biological activity of the cells using this technology; inject these transfected cells with enhanced potency back into the patient; and monitor response.

“Although electroporation has been around for quite a while, we have developed a scalable method that can electroporate as few as half a million or up to 10 billion cells at one time in less than 30 minutes,” said Dr. Brady. “We sell our system to pharmaceutical companies that want to generate batches of cells for screening specific target molecules or specific reporters, and they want to avoid all the time and effort that is needed to make a stable cell line or to use an expensive transfection reagent.” 

At the meeting, Dr. Brady and his colleagues provided data that was generated by some of MaxCyte’s customers showing that the technology has been used to screen ion channels, GPCRs, and other targets.

“This technology works with a variety of critical targets and a variety of important cell types. It gives you assay results that are comparable to that in stable cell lines, and it can generate the cells much more quickly,” Dr. Brady concluded.

Luminescence Detects Efficiency

As in many transfection techniques, electroporation efficiency is easily assessed by transfecting a reporter molecule into the cell. This method is especially effective in difficult-to-transfect cells (e.g., primary cells) where transfection efficiencies can be in the single-digit percentages.

For example, it was recently reported that transient transfection of CHO cells with PerkinElmer’s aequorin platform is a useful method to set up high-throughput screening, said Vincent Dupriez, Ph.D., principal scientist, who added that the Amaxa Nucleofector technology from Lonza has allowed for the transfection in primary cells.

“With the Amaxa Nucleofector technology, DNA is driven directly into the cell’s nucleus and the time from transfection to translation is shorter compared to other technologies, resulting in protein expression that can be observed within a few hours of transfection. This enables us to achieve rapidly a good level of aequorin expression in a large percentage of cells and to take advantage of the high signal-to-background ratio provided by the aequorin technology,” Dr. Dupriez explained.

The data discussed was derived from the transfection of two types of primary cells—human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells from the lung (HMVEC-L) cells. “Aequorin plasmid DNA was transfected into these two cells types, and then we performed sets of aequorin assays,” he added.

“The Amaxa transfection technology now enables using the aequorin technology in primary cells, providing a new pharmacological tool to study endogenous receptors in a natural cellular environment for labs equipped with tabletop-size or larger luminometers,” he continued.

“As an example, subtype-specific histamine receptor agonists and antagonists were used to demonstrate that the histamine receptor subtype 1 is the one responsible for the calcium response to histamine in both cell types tested.

This opens new possibilities for the characterization of the repertoire of receptors functionally present in primary cells and for the characterization of newly developed molecules in physiologically meaningful cellular models,” he concluded. 

In her talk at SBS, Nicole Faust, Ph.D., director of R&D at Lonza Cologne, presented data on how primary cells can be used in cell-based assays. She explained that one prerequisite for many cell-based assays is the efficient genetic manipulation of the cells in order to introduce reporter genes or biosensors for second messengers.

“Using the Amaxa Nucleofector technology, we successfully introduced the Ca2+-sensitive photoprotein aequorin into a variety of primary cells and obtained robust signals in response to the appropriate stimuli,” explained Dr. Faust.

“Using these modified primary cells we could obtain pharmacologically relevant dose-response curves for a number of endogenously expressed target proteins.

“So far, use of Ca2+-sensitive photoproteins has been mainly restricted to recombinant cell lines,” she continued. “By transferring this technology to human primary cells, we enable researchers to assay endogenous receptors or other drug targets within their natural context in a relevant cell background.”

Chemical Reagents

“The trend in transfection, as we see it, is that it’s moving increasingly toward more hard-to-transfect cell lines,” said Laura Juckem, Ph.D., R&D group leader at Mirus Bio. “We are finding that researchers want to be in more physiological cell types, such as primary cells, which are more relevant to the disease or process that they are studying.”

In response to this need, the company developed a reagent called TransIT-2020, which is designed to transfect cells typically recalcitrant to transfection such as primary cells.

These transfection technologies are based on different platforms depending on the type of nucleic acid to be transfected. “We found through extensive testing of our compound libraries that there are different thresholds of performance within our reagents, and some are better suited for use with a particular nucleic acid,” said Dr. Juckem. For example, she added, “We have a totally different reagent for messenger RNA than for other nucleic acids. It is the only one that is commercially available to deliver messenger RNA in the presence of serum.”

Dr. Juckem’s presentation at “Bioprocess International” later this month will focus on maximizing protein yield in suspension CHO cells. “Efficient transient transfection methods will allow researchers to produce high titers of proteins during preclinical drug discovery testing while the stable cell lines are under development,” Dr. Juckem remarked.

High-performance plasmid transfection: Primary human small epithelial cells were transfected using Mirus Bio’s TransIT-2020 and an EGFP expression plasmid (4:1 reagent-to-DNA ratio). Images were taken 24 hours post-transfection using a Zeiss axiovert inverted fluorescence microscope.

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