For in vitro mRNA transfection, jetMESSENGER® is our reagent of choice. Indeed, jetMESSENGER® protocol is simple: the reagent is ready-to-use and is compatible with the standard growth media (with or without serum and antibiotics) of a wide variety of adherent and suspension cell types. jetMESSENGER® operates through a very gentle process and upholds cell viability and overall cellular morphology in a variety of hard to transfect cells, such as rat cortex neurons, human liver carcinoma cells and mouse embryonic stem cells.
For in vivo mRNA delivery, in vivo-jetRNA®+ is our best-in-class reagent. in vivo-jetRNA®+ is a ready-to-use transfection reagent composed of preformed liposomes specifically developed to deliver mRNA in vivo. This reagent can be used to target unique or multiple organs, by using systemic injection routes, in various animal models (mice, rat, etc.). mRNA delivery using in vivo-jetRNA®+ is user-friendly, with a simple 2-step protocol and is highly efficient with comparable delivery results as Lipid Nanoparticles (LNPs). in vivo-jetRNA®+ can be used for vaccination/immunization purposes, anti-cancer studies, genome editing using CRISPR/Cas9 method or protein replacement.
With the LipidBrick® family, we also offer a range of cationic lipids dedicated to the formulation of lipid nanoparticles (LNPs). These active lipids protect the mRNA molecules and play an important role in the transfection capacity of LNPs. Importantly, by being based on an imidazolium polar head, LipidBrick® broadens the spectrum of current LNP applications in terms of potency and biodistribution by adding an overall positive charge to LNPs: this translates into greater delivery of mRNA to the lungs and spleen while reducing accumulation in the liver compared to LNPs based on ionisable lipids. Furthermore, within the LipidBrick® family, LipidBrick® IM21.7c is the cationic lipid (active lipid) used in the formulation of jetMESSENGER® and in vivo-jetRNA®+, allowing a seamless transition between our ready-to-use reagents and your LNP formulation tailored to your needs and applications.
mRNA can be in vitro transcribed (IVT) from DNA template using commercially available solutions (e.g. HiScribe™ kit provided by New England Biolabs) or be purchased “custom made” through different oligonucleotide suppliers such as Trilink, Eurofins, Aldevron, etc.
Of note, DNA template template must be linearized and contain a RNA polymerase promoter region such as T7 or SP6 upstream of the sequence to be transcribed, in 5’ for the sense RNA or in 3’ for the antisense RNA. If required, the RNA polymerase promoter region can be added upstream of the sequence to be transcribed by PCR using primers containing the minimal promoter sequences.
Learn more about mRNA synthesis.
Generally speaking, mRNA purification following in vitro transcription is not necessary, as there should be minimal RNA contaminants. If needed, several methods are suitable, including Phenol-chloroform Extraction/Ethanol Precipitation, LiCl precipitation, gel purification methods, and several commercially available mRNA isolation kits.
There is no limit in mRNA size per se. It is worth to keep in mind that mRNA synthesis kits are usually optimized for mRNA transcripts ranging in size from 0.3 to 5kb, equivalent to a protein size of 10-150 kDa. Therefore, for shorter or longer mRNA transcripts, additional optimization may be required to obtain a similar high yield.
In comparison to DNA transfection, mRNA-mediated transfection with jetMESSENGER® and in vivo–jetRNA®+ enables a more controlled gene expression that is not promoter dependent, over a similar period of time. In actively dividing cells, mRNA expression will be more prominently and stably observed between 1 to 4 days post-transfection. In slow to non-dividing cells, mRNA expression can be stable over a longer period of time, as for example up to 7 days in primary neuronal cells.
During the synthesis of endogenous eukaryotic mRNA, a 7-methylguanosine CAP is added in 5’ on the nascent transcript to protect it from degradation and increase translation rate. The presence of a polyA-tail in 3’ of nascent mRNA further protects from degradation and plays a role in the nuclear export of the mature mRNA. Therefore, existing mRNA synthesis kits usually offer solutions to improve stability of mRNAs by incorporation of 5’ cap analog, and 3’ polyA-tail. In addition, specific base modifications within the mRNA sequence such as 5-methylcytosine and/or N6-methyladenine can be incorporated for increasing stability and decreasing immunogenicity. Custom-made mRNA ordered from an oligonucleotide provider can be ordered with specific modifications such as Pseudo-Uridine for increased stability.
Increasing the stability of the RNA transcript may not always be necessary. For example, some viral RNA transcripts do not need to be capped and polyadenylated in order to start replicating and/or produce infectious virus after transfection.
mRNA delivery is suited for transient expression, but not for the generation of stable clones. mRNA cannot integrate into the cell genome, which prohibits the generation of stable clones but has the advantage of not modifying the genome of the host cell.
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