Transfection (i.e., artificial delivery of nucleic acids into cells) is a critical step in many applications, including studies of gene function or gene editing. In addition, enhancements to transfection technology over time (in both in vivo and in vitro settings) have been pivotal to advancing clinical research.

Optimized transfection practices are key to the production of therapeutic proteins for biopharmaceutical purposes, and advancements in lipid nanoparticle technology have proven focal to vaccine research. In addition, cell and gene therapy research has relied on efficient and safe transfection technologies for the continued development of treatments for various diseases.

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Gene modification delivery

Transfection is a vital step in applications of gene modification, whether that be gene editing or modulation of gene expression.


Genome editing

Efficient transfection of CRISPR-Cas9 reagents is essential to successful gene editing experiments, which have led to a multitude of clinical research breakthroughs. These breakthroughs include the restoration of fetal hemoglobin expression in hematopoietic stem/progenitor cells (HSPCs) as a potential treatment for patients with beta thalassemia [1] and the engineering of immune cells (e.g., T cells) to target tumor cells in cancer patients [2]. Different Cas9 and gRNA formats are available for transfection, each with their own benefits and drawbacks to use.

Figure 1. Sample CRISPR-Cas9 workflow. A gRNA molecule targeting a certain gene is transfected into the cell along with a Cas9 nuclease. Cleavage of the gene is verified, and edited cells can then be analyzed and expanded.

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Gene expression using mRNA or plasmids

A very common application of transfection is the delivery of nucleic acids into cells for the purpose of expressing certain genes. Transfected plasmid DNA or RNA supports production of recombinant proteins, including proteins with detectable markers or other modifications. mRNA transfection has also become a popular application for vaccine studies. It is important to note that plasmid DNA must enter the nucleus prior to protein expression whereas direct transfection of mRNA allows this nuclear entry step to be skipped, making mRNA transfection the more simplistic option.

Figure 2. Sample gene expression workflows. Plasmid DNA or mRNA encoding a gene of interest can be transfected into cells to facilitate expression of certain proteins.

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Gene inhibition using siRNA, miRNA, or shRNA

RNAi has become pivotal to studies of gene function, allowing for accurate and effective knockdown of genes to better understand their cellular roles. RNAi molecules include siRNA, miRNA, and shRNA, all of which can be designed to bind target mRNA molecules, preventing translation and the expression of certain genes. RNAi has also been utilized in gene therapy studies, facilitating the manipulation of gene expression for clinical purposes.

Figure 3. Sample gene inhibition workflow. RNAi molecules designed to inhibit expression of targeted genes are transfected into host cells, where they inhibit mRNA translation.

Learn more about RNAi and non-coding RNA research
Learn more about selecting an RNAi strategy


Stable transfection

Stable transfection allows for the sustained presence and expression of transfected DNA in cells via the incorporation of the foreign DNA into the host genome. Subsequent selection of cells containing this DNA can be performed using an antibiotic, allowing for the establishment of a stable cell line. Although more laborious, this application allows for continued expression in cell lines without repeated transfections and is therefore useful in many research areas, including long-term genetic regulation studies, large-scale protein production, and gene therapy.

Figure 4. Sample stable transfection workflow. The transfected DNA is incorporated into the host cell genome, allowing for selection and expansion of cells to generate a stable cell line.

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Transient transfection

In transient transfection, the nucleic acid delivered into the cell is only present and expressed for a limited amount of time. This very common application is used in studies where temporary modifications to cells are desired, including mRNA transfections, many plasmid transfections, RNAi studies, and certain gene editing experiments. Compared to techniques involving stable expression, transient expression is simpler and less laborious to achieve.

Figure 5. Sample transient transfection workflow. Transfected nucleic acids are not integrated into the host genome and therefore remain in the cell for a limited amount of time.

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Co-transfection

Co-transfection involves the delivery of two separate nucleic acids simultaneously. This type of transfection is useful in a variety of experimental applications, such as transfection of a marker or selection gene along with another nucleic acid or transfection of multiple plasmids into packaging cells for viral production. In addition, this application can be used for the transfection of separate Cas9 nuclease and CRISPR gRNA vectors.

Figure 6. Sample co-transfection workflow. Two nucleic acid (e.g., a plasmid expressing a marker gene such as GFP and siRNA) are transfected concurrently into a host cell.

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In vivo transfection

In vivo transfection allows for the direct delivery of DNA or RNA into live animals for modification of gene expression in target tissues. This application of transfection has become a key component of many important clinical studies, including in vivo mRNA transfection for vaccine and cancer research.

The delivery of mRNA and other nucleic acids using lipid nanoparticles (LNPs) is also rapidly emerging as an efficient and effective means of transfection in both in vivo and ex vivo applications. LNPs are being leveraged for clinical research and have proven especially important to the development of vaccines, including those used against COVID-19. LNPs may also be modified for organ-specific delivery of payloads, making them highly valuable in the development of targeted in vivo treatments and are considered more advanced and less toxic than traditional cationic lipid-based methods [3].

Figure 7. Sample in vivo transfection workflows. Using specialized reagents and techniques, nucleic acid molecules are delivered directly into a live organism.

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Transfection for protein expression

Protein expression studies are vital to analyses of protein structure and function in the lab. In addition, efficient transfection for protein expression is key to large-scale protein production in biopharmaceutical or biotechnological settings. Proteins can also be modified for improved stability or tagged for purification and detection. To ensure success, transfection of genes encoding recombinant proteins must be done with high efficiency and low toxicity.

Figure 8. Sample protein expression workflow. A plasmid encoding a protein of interest is transfected into a cell and subsequently isolated for further study and use.

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Viral vector delivery

Viral vector delivery can be used for both transient and stable transfection and is effective in many cell types. The high transfection efficiencies of viral vectors and their opportunities for sustained gene expression also make them an attractive option for cell and gene therapy studies.

Figure 9. Sample viral vector delivery workflow. Viruses engineered to encode a gene of interest are transfected into cells that can then express the gene.

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Transfection for cell and gene therapy

Cell and gene therapy is a growing and rapidly evolving area of clinical research that relies on safe and efficient transfection technologies for cell and gene modulation. Lentiviral (LV) and adeno-associated viral (AAV) vector delivery are two well-established and popular methods for transfection in cell therapy. In addition, other methods such as electroporation have also been employed in clinical settings (see CTS Xenon Electroporation System). By correcting faulty genes or allowing the expression of vital proteins in individuals with certain diseases, gene therapy is a highly valuable tool for providing lasting treatment.

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Primary cell transfection for immunotherapy development

Many immunotherapies, including chimeric antigen receptor (CAR) T cells for cancer treatment, require delivery or modification of genes. Transfection into immune or primary cells requires higher quality transfection equipment and reagents to ensure reliable and efficient results.

Both the Neon system and Lipofectamine 3000 have been shown to deliver genes into primary cells with low toxicity and high efficiency.

Figure 10. Sample immunotherapy workflow. Immune cells are transfected for CRISPR editing or with a plasmid. Following genetic and protein verification, cells containing the desired gene are selected and expanded for therapeutic use.

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In any transfection application, highly efficient delivery can significantly accelerate time to results by eliminating labor-intensive and time-consuming selection steps. It is important to note that the method of transfection used should be optimized for your specific cell type and application.

Learn more about transfection methods

Thermo Fisher Scientific is ready to help you select the application of transfection that is right for your experiment.

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TransfectionSelect tool

Find products, citations, and protocols optimized for your transfection experiments. Input information on your experiment type, cell line, and payload to unlock solutions.


References

  1. Zakaria NA, Bahar R, Abdullah WZ, et al. Genetic Manipulation Strategies for β-Thalassemia: A Review. Front Pediatr. 2022 Jun 15;10:901605. doi: 10.3389/fped.2022.901605. PMID: 35783328; PMCID: PMC9240386.
  2. Baghini SS, Gardanova ZR, Abadi SAH, et al. CRISPR/Cas9 application in cancer therapy: a pioneering genome editing tool. Cell Mol Biol Lett. 2022 May 4;27(1):35. doi: 10.1186/s11658-022-00336-6. PMID: 35508982; PMCID: PMC9066929.
  3. Yan Y, Liu XY, Lu A, et al. Non-viral vectors for RNA delivery. J Control Release. 2022 Feb;342:241-279. doi: 10.1016/j.jconrel.2022.01.008. Epub 2022 Jan 10. PMID: 35016918; PMCID: PMC8743282.

For Research Use Only. Not for use in diagnostic procedures.

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