mRNA Transfection

mRNA Transfection


Transfection is a powerful technique used in research to modulate gene expression in eukaryotic cells both in vitro and in vivo. It consists of the delivery of exogenous nucleic acids through the cell membrane into cells. The most commonly used exogeneous nucleic acids used in transfection are plasmid DNA (pDNA) and messenger RNA (mRNA).

While DNA has been the most popular choice for a long time, mRNA transfection offers distinct benefits that make it useful for varied applications.

Oz Biosciences offers a wide range of mRNAs, both modified and unmodified, that can be used as gene reporters, as genome editing mRNAs or as control mRNAs for vaccines.

Why choosing mRNA over pDNA in transfection?

One of the principal barriers in pDNA transfection is the need to pass through the nuclear membrane for transcription to occur. Hence, when slow or non-dividing cells need to be transfected, the use of pDNA can make this process a very lengthy one. The use of mRNA, on the other hand, does not require nuclear uptake for expression since translation of mRNA occurs in the cytoplasm. Therefore, protein expression can occur at a much faster pace when mRNA is used as the protein is directly expressed in the cytoplasm.

Furthermore, mRNA transfection is cell cycle-independent, which makes mRNA transfection even more attractive for slow-dividing cells such as endothelial cells or dendritic cells.

Another benefit of using mRNA in transfection is the reduced risk of genomic integration as, contrary to pDNA, mRNA cannot be inserted into the target cell’s genome, avoiding unwanted mutations.

Finally, protein expression occurs in a totally promoter-independent manner and since mRNA-based protein expression is sustained for a limited time only, this is a transient process that can also be of benefit for certain applications.

OZ Biosciences offers mRNAs that mimic fully processed mature mRNAs. These mRNAs are stabilised with 5’ Cap 1 structure and 3’ poly(A) tail and are optimised to yield improved stability and performance. They offer unmodified and modified mRNAs (in which uridine has been replaced by which 5-methoxyuridine, or 5moU, to reduce innate immune responses).

mRNA structure

5’ Cap This cap structure protects mRNA from degradation and recruits processing and translation factors. In mammals, the predominant form is a 7-methyl-guanosine (Cap 0) linked via a 5’ to 5’ triphosphate bridge to the first transcribed nucleotide which is methylated on the ribose O-2 position (Cap 1). Other methylations are also observed including first transcribed nucleotide adenosine methylation on position 6 and second transcribed nucleotide ribose O-2 methylation.

5’ Untranslated Region (5’ UTR) The 5’ UTR is a non-coding region directly upstream from the initiation codon involved in the posttranscriptional regulation of gene expression by modulating mRNA stability, transport, subcellular localisation and translation efficiency thus allowing fine control of the protein product. This region has a high GC content and several secondary structures and comprises the Kozak sequence (GCCGCCRCCAUGG) that plays a major role in the initiation of the translation process.

Open Reading Frame (ORF) This internal region of eukaryotic mRNA is translated into protein. The ORF begins with a methionine codon (AUG) and ends with a stop codon.

3’ Untranslated Region (3’UTR) The 3’ UTR is the part of mRNA that immediately follows the translation termination codon. This region plays a crucial role in gene expression by influencing the localisation, stability, export, and translation efficiency of an mRNA. It contains various sequences including microRNA response elements (MREs), AU-rich elements (AREs), and the poly(A) tail.

Poly(A) tail The poly(A) tail is a long sequence of adenine nucleotides (0-250 nucleotides with a median length of 50-100 in HeLa and NIH-3T3 cells)2 added to the 3’ end of the pre-mRNA. The poly(A) tail contains binding sites for poly(A) binding proteins (PABPs) that play a major role in export from the nucleus, translation, and protection from degradation. Its length is an important determinant of translational efficiency and mRNA stability. This is an important element of a mRNA as its absence or removal often leads to exonuclease-mediated degradation of the mRNA.

mRNA categories

Oz Biosciences offers three categories of mRNAs:

Reporter gene mRNAs

Ideal controls to study transfection efficiency.

Reporter genes are commonly used in cell biology research. Reporter mRNA can be used as controls to study transfection and expression in mammalian cells using a variety of assays. These capped (Cap 1) and polyadenylated mRNAs are optimized for mammalian systems and are composed of unmodified or modified (moU replaces U) nucleotides, and they mimic fully processed mature mRNA.

Reporter Genes mRNADescription
GFP mRNA (modified and unmodfied)
Designed to produce high expression level of Green Fluorescent Protein. It is a commonly used direct detection reporter in mammalian cell culture, yielding bright green fluorescence with an excitation peak at 488 nm and an emission peak at 507 nm
F-Luc mRNA (modified and unmodified)
Designed to produce high expression level of FireFly Luciferase. It is commonly used in mammalian cell culture to measure both gene expression and cell viability. FireFly Luciferase emits bioluminescence in the presence of the substrate, luciferin.
mCherry mRNA (modified and unmodified)
Encodes the mCherry fluorescent protein which is derived from DsRed, a protein found in Discosoma sp. mCherry is a monomeric fluorophore with a excitation peak at 587 nm and emission at 610 nm. mCherry is photostable and resistant to photobleaching.
Beta-Gal mRNA (modified and unmodified)
Encodes for a protein product of the bacterial LacZ gene. Beta-Gal catalyzes the conversion of Beta-galactosides into monosaccharides. It is a common marker gene used to assess transfection efficiency
Tomato mRNA (modified and unmodified)
Designed to produce high expression level of Orange Fluorescent Protein. This mRNA is produced by in vitro transcription and stabilized at the 5’ end by modified nucleotides capping.

Genome editing mRNA

Cas9 mRNA for CRISPR Genome editing

“Genome editing” or “Genome engineering” gives the ability to introduce a variety of genetic alterations (deletion, insertion…) into mammalians cells. During the past decade, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were the tools of choice for genome editing technologies until the recent discovery of CRISPR/Cas9 technology that has revolutionized the field. Plasmids and viral vectors are traditionally used in genome editing to express the required proteins. However, there is a risk to use DNA instead of mRNA:

  • Cas9: Double-stranded DNA breaks catalyse the insertion of DNA at the cut site. At some substantial frequency, the protein expression vectors can integrate, which can lead to continuous expression of the nuclease or a previously silent sequence.
  • Cre Recombinase: Site-specific recombinases are useful tools for the manipulation of genomes. However, continued expression of a recombinase in a cell or in vivo can result in toxicity and undesired off-target recombination. For this reason, transient expression from mRNA is an ideal method for recombinase expression.

Hence, mRNA is becoming a powerful tool in genome editing. With no risk of insertional mutagenesis, mRNA is used to transiently express the required proteins. Wild-type Cas9 mRNA is a stabilised non-immunogenic messenger RNA (mRNA) that has been designed to produce a high expression level of wild-type Cas9 protein. It creates a double-stranded break at a target site delineated by RNA guide sequences.

This mRNA is produced by in vitro transcription and is optimized to yield improved stability and performance and to lower the cellular innate immunity response thanks to the introduction of the 5’ Cap and the 3’ Poly(A) tail end.

Gene Editing mRNADescription
Cas9 endonuclease mRNA (modified and unmodified)
The RNA-guided Cas9 endonuclease is used to induce site-directed double-strand breaks in DNA. These breaks can lead to gene inactivation or introduction of heterologous genes, providing an efficient tool for Genome Editing.1-4
CRE Recombinase mRNA (modified and unmodified)
Site-specific DNA recombinases are widely used in cells and organisms to manipulate the structure of genomes and to control gene expression by targeted activation or de-activation. Each recombinase catalyzes 4 types of DNA exchange reactions (figure 4) between short specific target sequences (30-40 nucleotides).
  1. Cong L. et al, . Science. 2013;339 (6121):819-823.
  2. Mali P. et al, Science. 2013;339 (6121):823-826.
  3. Jinek M. et al, Science. 2012;337 (6096):816-821.
  4. Cho SW. et al, Nat Biotechnol. 2013;31(3):230-232.

Vaccine mRNAs

Ideal as controls for immunisation or vaccine studies.

A nucleic acid-based vaccine combines the positive features of live attenuated vaccines while avoiding many potential safety limitations.1 These vaccines present several advantages over conventional vaccines such as:

  • Mimicking a live infection by expressing antigens in situ after immunisation and priming both B and T cell responses including cytotoxic T lymphocytes.2
  • Revealing focused immune responses directed toward the selected antigens of interest with no potential reversion to pathogenicity.

Furthermore, mRNA vaccines present a better safety profile than DNA vaccines: DNA vaccines display a long-term expression, a potential risk for genome integration and induction of anti-DNA antibodies.3

All the advantages of mRNA vaccines come from the intrinsic properties of mRNA:

  • They are produced using cell-free enzymatic transcription.
  • The transient expression of mRNA encoded antigen enables a more controlled antigen expression and minimises the risk of tolerance induction that can be associated with long-term exposure.4
  • There is thus an absence of any additional encoded protein which exclude the possibility of raising undesired immune response or interaction with the host.5
  • Their stabilised design allows a higher level of expression in vivo.6
  • mRNA serves the dual purpose of expressing the desired antigen as well as acting as an adjuvant.
  • mRNA has a superior safety profile compared to inactivated viruses or pathogens.

Oz Biosciences offer modified OVA mRNA (5moU) and unmodified OVA mRNAs, produced by in vitro transcription and stabilised with the 5’ Cap and the 3’ Poly(A) tail end. OVA mRNA encodes for OVA protein, a commonly used antigen for immunisation and biochemical studies and also an established model allergen for airway hyper-responsiveness. OVA mRNAs (5moU) resemble fully matured mRNAs and are ready to be translated by the ribosome.

It is important to notice that exogenous unmodified mRNAs activate the innate immune system and cytokines production in order to influence induced immune response, but these mRNAs are rapidly degraded. The nucleoside modification in modified OVA mRNAs reduces the immune effect compared to unmodified mRNA but lasts longer in the organism due to the stabilisation and protection that the modification confers to the mRNA.


  1. Deering RP et al, Expert Opin Drug Deliv. 2014. Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines.
  2. Johansson DX et al, PLoS One. 2012. Intradermal electroporation of naked replicon RNA elicits strong immune responses.
  3. Pascolo S., Handb Exp Pharmacol. 2008. Vaccination with messenger RNA (mRNA).
  4. Pollard C. et al, Trends Mol Med. 2013. Challenges and advances towards the rational design of mRNA vaccines.
  5. Schlake T. et al, RNA Biol. 2012. Developing mRNA-vaccine technologies.
  6. Kallen KJ. et al, Hum Vaccin Immunother. 2013. A novel, disruptive vaccination technology: self-adjuvanted RNActive(®) vaccines.

OZ Biosciences mRNAs for Vaccination/Immunisation:

mRNA for vaccinesDescription
OVA mRNA (Unmodified)
Activates innate immune system and production of cytokines in order to engage induced immune response but mRNAs are rapidly degraded
OVA mRNA (moU)
Increased stability and translational capacity of mRNA while diminishing its immunogenicity in vivo

Furthermore, OZ Biosciences has recently introduced Spike SARS CoV-2 (E484K; N501Y) mRNA to their portfolio. This mRNA encodes for the Spike protein of the SARS-CoV-2 virus and bears two mutations (E484K and N501Y). Spike protein is used as antigen for immunization and biochemical studies. For Research Use Only. Not for use in humans. Not for use in diagnostic or therapeutic purposes. Spike mRNAs resemble fully matured mRNAs with 5’cap1 structure and 3’ polyA tail, therefore ready to be translated by the ribosome. Two versions are available, mRNA-34, which does not bear any additional nucleotide modifications, while mRNA-35 is modified with 5-methoxyuridine (5moU) to reduce innate immune responses.

Spike protein SARS-CoV-2 mRNADescription
mRNA34 (unmodified)Spike protein is used as antigen for immunization and biochemical studies.
mRNA35 (5moU)Modified with 5-methoxyuridine (5moU) to reduce innate immune responses.

To find out more about these products and how they could help you with your research, don’t hesitate to get in contact with one of our tech support members at

mRNA Transfection
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