Blogs Transient Transfection: The Pragmatic Alternative to using Stably Transfected Cells for Luciferase Reporter Assay High Throughput Screening

Transient Transfection: The Pragmatic Alternative to using Stably Transfected Cells for Luciferase Reporter Assay High Throughput Screening

Transient Transfection blog (3)

Cell-based Luciferase reporter assays are commonly used to study gene expression, signaling pathways, and transcriptional regulation. Reporter cells are engineered to over-express a receptor of interest and a luciferase reporter gene, often on a second vector, that is responsive to the activation-status of that receptor. There are two main types of cells used in luciferase reporter assays: stably transfected cells and transiently transfected cells. Unfortunately, there is a broadly held misconception that using stably transfected cell lines are the superior, or only, choice when contemplating high-throughput screening initiatives. Before using a stably transfected cell line for long-term screening projects, it is important to understand their pitfalls which may lead to longer project times, higher costs, and assay performance drift.

The Nuts-and-Bolts of Stable Cell Lines

Stable cell lines are generated by transfecting (or transducing) cells with vectors that drive the expression of a target receptor, as well as a luciferase gene that is functionally linked to upstream genetic response elements that are specific to that receptor. The population of transfected cells are then cultured under long-term selective pressure, allowing the isolation and propagation of those relatively few cells that have integrated copies of the introduced plasmids or viral vectors. This initial process of generating stable cell lines is both arduous and expensive, requiring significant time and effort to screen primary clones to identify the few with acceptable assay performance characteristics. Namely, clones in which the integrated receptor gene is constitutively expressed at high levels, and the luciferase reporter gene is transcriptionally silent in the absence of receptor activation, but capable of rapid, high-level induction upon receptor activation.

It’s true that stably transfected reporter cell lines are commercially available for many assays and simply purchasing them is preferred. However, acquiring commercial division-competent cells typically involves an exorbitant up-front fee and often requires annual licensing fees for utilizing the technology. A different iteration of these commercially available cells is “division arrested” cells. The fees to use them are relatively moderate and their use does not require a licensing agreement, but the cells cannot be expanded or banked. Unfortunately, because they are division arrested, the cells are committing to apoptosis. Particularly in the case of running antagonist-mode assays, some may find concern in using reporter cells that are in the process of dying.

The Paradox of “Stable” Cell Lines: Genetic Drift

One misperception about using stably transfected cell lines is the belief that a clone can be maintained in selective culture, expanded exponentially, then banked as a homogenous population of reporter cells. The dogma is that once selected, performance of the reporter cell line is ‘locked in’, capable of delivering an unwavering and robust functional response in all future assay setups. Unfortunately, this is not true. Regardless of whether the clone was generated in one’s own lab or obtained as a commercial product, “Stable cell lines” are not at all stable (Indeed, the term is in league with “jumbo shrimp” as being the perfect oxymoron).

Proof of the inherent genetic instability of these cells is the requirement to propagate them under continuous chemical selection. Engineered cell lines that have integrated multiple copies of foreign DNA are particularly susceptible to genetic drift. This is due to a variety of factors, but it is most commonly the result of rearrangement, homologous recombination, or excision of unstable sequence blocks during cell division. Integrated exogenous vectors are also subject to shifting patterns of epigenetic regulation. Such genetic restructuring events can significantly alter expression and function of the receptor gene and/or the luciferase reporter gene resulting in degraded or lost luciferase reporter assay performance. Genetic drift happens on an individual cell basis, not a population basis. And genetic rearrangements that otherwise crush luciferase reporter function may yet retain expression of the selective marker genes. Hence, individual cells with no, or low, reporter function may thrive under selection, leading to the emergence of subpopulations within a presumed homogenous cell line.

Continuous low-level genetic drift within an original clone is impossible to manage in high throughput screening (HTS) and it complicates the correlation of assay results that were obtained from earlier passages of the reporter cells, or from new ‘thaws’ of cryopreserved cells. To minimize the effects of genetic drift, time and resources must be expended to periodically interrogate clonal lines for changes in receptor / luciferase reporter gene functions. Long-term cultures that show degraded assay performance should be discarded, and a new ‘split’ of the early-passage clone awakened from cryo-storage.

Overcoming an Irrational Bias: The use of Transiently Transfected Reporter Cells

Transiently transfected reporter cells, which harbor non-integrated forms of the receptor expression vector and the luciferase reporter vector, provide many technical advantages over genome-integrated vectors. The most obvious contrast between the two is the amount of time and resource expenditure required. When generating stably integrated reporter cell lines months of work are required to select and expand primary clones, followed by weeks assessing the performance of those individual clonal lines. By comparison, the construction of reporter cells using transient transfection methods is ultra-fast, requiring a week of work.

More importantly from a scientific standpoint, transient transfection methods allow researchers great flexibility to fine-tune transfection conditions to achieve highly optimized reporter cell performance. Both the cell type used and composition of the transfection cocktail will greatly influence the final assay metrics. Achieving maximum RLU (relative light units) signal, minimum RLU background, high values of fold-activation or fold-reduction, and low EC50 and/or IC50 values are possible by performing small-scale experiments to optimize key variables. Researchers may easily and quickly investigate the impact of using several different host cell types, transfection reagents, and [receptor vector : Luciferase vector : transfection reagent] ratios. Evaluating the co-introduction of vectors that express an accessory co-activator, co-repressor, co-receptor, or even specific transporter is easy and frequently is beneficial to final assay performance.

The ability to fine-tune reporter cell performance through transient transfection methods is in stark contrast to that when generating stable cell lines. Good or bad, all one gets is the performance of the best clone selected. There is no opportunity for iterative rounds of reporter cell optimization, so one can only hope that the performance of the best stable clone is sufficiently robust for HTS applications.

A common, but unfounded, concern about using transiently transfected reporter cells is the perceived risk of lot-to-lot variability. This discounts the fact that transient transfections can be performed reproducibly and at any scale. Commercial providers, such as INDIGO Biosciences, routinely manufacture reporter cells at a scale of approximately 80-100 tubes of cryopreserved cells, each tube being sufficient to seed one 384-well assay plate. Scaling up to manufacture several thousand tubes of reporter cells is easily achievable, and often done. In this way, a researcher’s HTS campaign can be completed using a single large lot of reporter cells, thus eliminating lot-to-lot bias.

 

INDIGO’s Approach to Luciferase Reporter Cell Assays

As a leading provider of cell-based luciferase reporter assay kits and screening services, INDIGO Biosciences exclusively uses transiently transfected reporter cells. A multitude of transfection variables are interrogated to ultimately yield highly optimized reporter cell performance. Standard Manufacturing Protocols are validated for large- and ultra large-scale manufacturing of reporter cells, thus ensuring high lot-to-lot reproducibility of assay metrics. INDIGO’s single use reporter cells are division competent, but there is no requirement for costly licensing agreements.

By employing transient transfection in our reporter assays, researchers in academia, environmental monitoring, pharmaceuticals, and biotechnology can consistently get high-quality, reliable, and reproducible data. Our team of experienced scientists are committed to offering quality assays and achieving customer satisfaction. We strive to make it easier than ever to obtain high-quality data that advance drug development, and an understanding of the mechanisms that underlie disease and environmental toxicity.