An Overview of Transfection Methods

This article presents the state-of-the art of transfection methods from their working principles to a detailed comparison of each technique with its pros and cons. With this overview, we will introduce the future of transfection methods: the FluidFM technology that can be employed for precision gene editing by direct intra-nuclear injection.


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What is the principle of transfection in biology? 

In brief, it's the process of delivering genetic information by inserting foreign nucleic acids in a cell 

An overview of transfection methods by Cytosurge

Transfection is a widespread cell culture technique and an effective tool to analyze gene functions and gene products in cells. Transfection describes the mechanism in which external nucleic acids are delivered into a eukaryotic cell to modify the host cell’s genome. [1,2,3] More precisely, in-vitro transfection refers to the delivery of cargo molecules into cultured cells whereas in-vivo transfection corresponds to the delivery of cargo molecules to the target tissue.


The success of such technique resides in the delivery efficiency of the genetic material into cells. The delivery efficiency is dependent upon the genetic material quantity and quality, the incubation time and the ratio of transfection reagent to DNA. Parameters such as the origin, type and the passage of transfected cells, and the presence or absence of serum in the cell culture are also key to make a successful genetic delivery. [4]


What is transfection used for? The applications.

Biological research

Gene and cell therapy

Bioprocessing

Agriculture & Plant Biotechnology

Do you need more information about transfection methods?

What are the two types of transfections?

Stable and Transient transfections!

Transfection can be classified into two types, namely stable and transient transfections [5,6].

Stable transfection refers to sustaining long-term expression of a transgene by integrating foreign DNA into the host nuclear genome or maintaining an episomal vector in the host nucleus as an extra-chromosomal element [7].

Oppositely, transient transfection does not require integrating nucleic acids into the host cell genome. [8] The latter is commonly used for short term expression of a desired gene that lasts a few days. Good examples of applications include research of gene expression, gene silencing studies and analysis of recombinant proteins. 

There are also two types of approach that can be defined as "chemical" and "physical". Chemical transfections are widespread methods thanks to the ease, cost, and broad choice of transfection reagents available on the market.

Chemical transfections comprise a number of steps from the plating of the cells at sub-confluency, preparing the transfection reagent / DNA complex immediately before transfection, to the assessment of the construct expression.

An overview of transfection methods by Cytosurge


Do you want full control over your single-cell manipulation?

What are the different methods of transfection?

Lipofection, Electroporation, Viral transduction, Co-Transfection, Calcium Phosphate Co-precipitation, DEAE-Dextran Transfection, MicroInjection... the list is long!

Lipofection - a standard chemical transfection - represents the transfection using liposomes, small molecules which can fuse with cell membranes and are able to release their contents. Common applications comprise research in Oncology notably to target specific genes (oncogenes) via small molecule-based drugs or gene silencing using RNAi technology.


Electroporation refers to a mechanical transfection where an electrical pulse is applied through an electroporation cuvette to create temporary pores in the cell membrane. The electrical pulse applied and the effectiveness of the technique itself depends on DNA concentration and the type of cells studied. 


Viral transduction also known as virus-mediated transfection describes the use of viral vectors to transfer nucleic acids into the cells. In the case of a CRISPR viral transduction, once the cas9 virus enters the cells, the CRISPR-cas9 system alters the cellular DNA. Afterwards, the transgenic host carry on the genetic modification expression. [9-10] 

An overview of transfection methods by Cytosurge


Co-transfection corresponds to the simultaneous transfection of two separate nucleic acids molecules such as plasmid DNA and siRNA


Calcium Phosphate Co-precipitation describes the process of mixing DNA with calcium chloride in a buffered saline/phosphate solution to generate a calcium-phosphate–DNA co-precipitate, which is then dispersed onto cultured cells. Calcium phosphate facilitates the binding of the condensed DNA in the co-precipitate to the cell surface, and the DNA enters the cell by endocytosis. The process efficiency is limited by a number of parameters including small changes in pH, temperature, and buffer salt concentrations. [11]


DEAE-Dextran Transfection employs the diethylaminoethyl-dextran (DEAE-dextran) - a polycationic derivative of dextran (a carbohydrate polymer) to facilitate transfection. When mixed with the DNA, the resulting complex is carried into contact with the negatively charged plasma membrane by the excessive positive charge provided by the polymer. 


Microinjection is commonly used to introduce DNA and RNA into single cells. With the aid a micromanipulator and microscope, the DNA or RNA is directly inserted into the cytoplasm or nucleus. This is typically time consuming, but results in very high transfection efficiency. A sub-category of injection called "nanoinjection" is enabled with the FluidFM Technology.


Due to the gentleness of the technique, the term "direct intra-nuclear injection" was used, and the expression "single-cell biopsy" was developed to describe the use of the same approach to perform extraction of genetic information from the cell without damaging the cell. Electroporation and cell microinjection are two good examples of physical transfection methods that are harsh on cells due to disruption of the cellular membrane and often results in cell death. Those are therefore inappropriate options for cells that have been traditionally difficult to transfect, unlike the method of the direct intra-nuclear injection.


Benefits of Direct Intra-Nuclear Injection

Precision delivery

based on an ultra-gentle transfection method based on nano-injection.

High CRISPR HDR Efficiency

Easily introduce multiple gene edits in one go while maintaining your cells alive. Fasten your stable monoclonal cell lines generation!  

High Cell Viability

The gentle injection or extraction ensure your cell viability. Especially suitable for hard-to-transfect cells and rare cell types.  

How to choose the right transfection method for my application? 

Transfection can be highly specific to the cell-type being transfected. Some cells are more sensitive to transfection while others are not. Thus, when defining a transfection experiment, it is particularly important to understand the final goal of the transfection; stable expression requires a different setup than does transient transfection, and as such, experimental protocols should be adjusted. If time is pressing, then transfection experiments can be outsourced to reliable companies that can guarantee the development of stable or transient cell lines for research applications. Being researchers ourselves, our team has identified and developed over the years and the research projects, an expertise in direct intra-nuclear injection.


Traditional Transfection Methods vs. Direct Intra-nuclear Injection


When millions of edited cells are needed, batch transfection methods are more appropriate. For example, lentiviral vectors are the method of choice when performing CRISPR genetic screens, due to the very high efficiency of transfection. In gene/cell therapy, electroporation would be preferred as viral vector often come with random insertion into the genome, which could bring undesirable effect when used in a patient. Injection methods are particularly appropriate when few modified cells (several hundreds) are needed. This is the case for example when creating CRISPR KO/KI monoclonal cell line, when working with rare cells or when multiplex editing is needed.


Do you want an unlimited access to reliable and reproducible monoclonal cell lines

The gene editing tool to perform direct intra-nuclear injection: The FluidFM® 

To address those challenges, Cytosurge employed its proprietary patented technology - the FluidFM technology - to offer a unique tool to perform direct intra-nuclear injection - the FluidFM OMNIUM. The FluidFM technology offers a unique in-vitro solution to improve the efficiency and applicability of CRISPR across a variety of cell types and for cell line development.



LipofectionElectroporationViral TransductionMicroinjectionFluidFM Nanoinjection
Hard-to-transfect cells






Multiplex Editing






HDR - Large knock-in






Genetic screens






Autologous gene / cell therapy






Allogeneic cell therapy (off-the-shelf)






Transfection of millions of standard cells






Generation of animal models







Table 1: Comparison of traditional transfection methods versus the FluidFM Nanoinjection - direct intra-nuclear delivery method. 




Not recommended


Works but with drawbacks


Works but not optimal in every situation


Method of choice

Conclusions - Take Away Messages

Adapt the transfection method to the cell type

Direct Intra-nuclear delivery for Multiplex Gene Editing and hard-to transfect cells

FluidFM - the only tool for direct single-cell intra-nuclear injection, extraction and manipulation.

References

[1] Chong, Zhi Xiong, Swee Keong Yeap, and Wan Yong Ho. "Transfection types, methods and strategies: A technical review." PeerJ 9 (2021): e11165.

[2] Kim & Eberwine (2010) Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. 2010;397(8):3173–3178. doi: 10.1007/s00216-010-3821-6.

[3] Chow et al. (2016) Chow YT, Chen S, Wang R, Liu C, Kong C-W, Li RA, Cheng SH, Sun D. Single cell transfection through precise microinjection with quantitatively controlled injection volumes. Scientific Reports. 2016;6(1):24127. doi: 10.1038/srep24127.

[4] Fus-Kujawa, Agnieszka, et al. "An overview of methods and tools for transfection of eukaryotic cells in vitro." Frontiers in Bioengineering and Biotechnology (2021): 634.

[5] Kim & Eberwine (2010) Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. 2010;397(8):3173–3178. doi: 10.1007/s00216-010-3821-6. 

[6] Stepanenko & Heng (2017) Stepanenko AA, Heng HH. Transient and stable vector transfection: pitfalls, off-target effects, artifacts. Mutation Research/Reviews in Mutation Research. 2017;773:91–103. doi: 10.1016/j.mrrev.2017.05.002. 

[7] Lufino, Edser & Wade-Martins (2008) Lufino MMP, Edser PAH, Wade-Martins R. Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression. Molecular Therapy. 2008;16(9):1525–1538. doi: 10.1038/mt.2008.156.

[8] Riedl et al. (2018) Riedl S, Kaiser P, Raup A, Synatschke C, Jérôme V, Freitag R. Non-viral transfection of human T lymphocytes. Processes. 2018;6(188):1–17. doi: 10.3390/pr6100188.

 [9] Glover, Dominic J., Hans J. Lipps, and David A. Jans. "Towards safe, non-viral therapeutic gene expression in humans." Nature Reviews Genetics 6.4 (2005): 299-310.

[10] Pfeifer, Alexander, and Inder M. Verma. "Gene therapy: promises and problems." Annual review of genomics and human genetics 2.1 (2001): 177-211.

[11] Graham, Frank L., and Alex J. Van Der Eb. "A new technique for the assay of infectivity of human adenovirus 5 DNA." virology 52.2 (1973): 456-467.