Transfection: Adding a Book to the Library

Part of the series: Biochemical Tools & Techniques

Cells contain a vast library of information that codes for every intricacy of life. What if we as biochemists could edit a page or insert an entirely new chapter? This is the underlying principle of transfection. It is the fundamental method that allows us to move from passive readers to active editors.

Figure 1. A biochemist adds a book to the library of genetic material.

Transfection, transduction, transformation: a crucial distinction

Before we dive into the toolkit, it is essential to define our terms. Transfection, transduction, and transformation all share the goal of introducing genetic material, but their mechanisms and applications are distinct.

1. Transformation: Introducing genetic material into bacterial or plant cells. (eg. Engineering E. coli to produce insulin)

2. Transduction: Using a viral vector to deliver genetic material to eukaryotic cells.

3. Transfection (Our focus): The non-viral introduction of new genetic material into eukaryotic cells. It is our most versatile, chemical-based tool for cell engineering.

The first critical choice

Transfection contains a broad family of strategies and vectors. Let's break it down a bit. As biochemists, we face the question: Would our experiment benefit from a long-term (stable) or short-term (transient) effect of the new genetic material?

• Stable transfection: The foreign DNA is integrated into the host's genome, creating a heritable change, passed on to all future daughter cells. (eg. genetically engineered cell lines)

• Transient transfection: The genetic material (DNA or RNA) does not become integrated in the host's genome and is not heritable. It is highly expressed for 24-96 hours before being diluted out as cells divide. It is for quick, response-based experiments.

Choosing your strategy

Suppose we wish to introduce heritable DNA into a cell line to measure cellular characteristics. In this case, we would choose stable transfection because we intend to integrate foreign DNA into the host's genome. Stable transfection has many positive attributes. However, there are drawbacks. Stable transfection is less efficient than transient transfection and can be more challenging.

In a separate experiment, we aim to induce a rapid and pronounced increase in the expression of a target protein. Using stable transfection may be sufficient to induce protein expression; however, a more effective approach is available. Transient transfection offers higher speed, greater flexibility, and higher throughput than stable transfection.

Consider the following: could we use RNA for stable transfection? No. Over time, ribonucleases degrade RNA, and protein expression does not persist for long. Now, how about transient transfection? Absolutely! RNA is highly effective for short-term induction of protein expression or gene silencing. The choice between stable and transient transfection depends on the intentions of your experiment.

Options for genetic material

Stable: DNA only

Transient: DNA or RNA

Here we have a few examples of when to use stable or transient transfection.

If your goal is...

• Quick functional test (reporter assay, siRNA knockdown) -> Transient

• High throughput screening (many conditions) -> Transient

• Creating a permanent cellular model (gene knockout line) -> Stable

• Biopharmaceutical production -> Stable

• Studying long-term adaptation or chronic effects -> Stable

Chong et al. (2021) synthesize the wide range of transfection methods and qualitatively score each. In Table 1, I synthesize their findings. Based on the experiment's objectives, a vector can be selected by weighing the characteristics of each technique.

Table 1. Vectors and their corresponding safety, efficiency, and cost/prep scores.

Sources:

Bastien Duckert, Steven Vinkx, Dries Braeken, Maarten Fauvart, Single-cell transfection technologies for cell therapies and gene editing, Journal of Controlled Release, Volume 330, 2021, Pages 963-975, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2020.10.068.

Chong, Z. X., Yeap, S. K., & Ho, W. Y. (2021). Transfection types, methods and strategies: a technical review. PeerJ, 9, e11165. https://doi.org/10.7717/peerj.11165