The Prodigious Potential of mRNA Electrotransfer as a Substitute to Conventional DNA-Based Transient Transfection

Abstract

Transient transfection of foreign DNA is the most widely used laboratory technique to study gene function and product. However, the transfection efficiency depends on many parameters, including DNA quantity and quality, transfection methods and target cell lines. Here, we describe the considerable advantage of mRNA electroporation compared to conventional DNA-based systems. Indeed, our methodology offers extremely high transfection efficiency up to 98% regardless of the cell line tested. Protein expression takes place a few hours post-transfection and lasts over 72 h, but overall, the electrotransfer of mRNAs enables the monitoring of the level of protein expressed by simply modulating the amount of mRNAs used. As a result, we successfully conducted cell imaging by matching the levels of expressed VHHs and the antigen present in the cell, preventing the necessity to remove the excess unbound VHHs. Altogether, our results demonstrate that mRNA electrotransfer could easily supplant the conventional DNA-based transient expression system.

Tracing endogenous proteins in living cells through electrotransfer of mRNA encoding chromobodies

“…We first evaluated the anti-nuclear lamin VHH binding to lamin A/C. [13] The nanobody coding sequence was cloned in frame with either the mScarlet, [20] the mScarlet fused to E3 peptide originated from the tetramerization domain of p53 [17] or the dTomato coding sequences [21] , the last two both forming dimers (Figure 1A). The DNA constructs were then used for IVT mRNA synthesis.…”

“…The mRNAs are synthetized in vitro and delivered into living cells by electroporation. [17] The possibility to precisely control the level of chromobody expressed by adjusting the amount of mRNAs transfected ensures the ability to fine-tune the level of chromobody with its cellular target. This was demonstrated by targeting three endogenous proteins of different abundance, namely β-actin, lamin A/C and Dnmt1.…”

“…Linearized plasmids were purified and used as a template for in vitro transcription (IVT) as previously described. [17] The transfection experiments with mRNAs were conducted with the NeonTM transfection system (Thermofisher Scientific, Waltham, MA, USA) as previously described. [17] The cells were transferred into 24-well plates containing glass coverslips or in Ibidi for time-lapse imaging.…”

“…Recently, we successfully detected endogenous proteins using electrotransfer of in vitro transcribed (IVT) mRNAs encoding chromobodies. [17] We have shown that mRNA is ideal for delivering genetic information into various cell lines with extremely high efficiency, up to 95%. Furthermore, by monitoring the amount of transfected mRNA, we were able to adjust the amount of nanobody expressed to the amount of target antigen inside the cell, eliminating the need to remove the excess of unbound nanobody.…”

“…Furthermore, by monitoring the amount of transfected mRNA, we were able to adjust the amount of nanobody expressed to the amount of target antigen inside the cell, eliminating the need to remove the excess of unbound nanobody. [17] In the present work, we describe the huge potential of mRNA encoding chromobody delivery for detecting and tracing intracellular targets with extremely high accuracy by live cell imaging. As a proof of concept, we tested three chromobodies to visualize the dynamic behavior of endogenous proteins present in different abundance and in distinct subcellular compartments, by real-time microscopy; the lamin A/C components of the nuclear lamina [13] , the β-actin, highly conserved protein and involved in various types of cell mobility [18] and the DNA methyltransferase 1 (Dnmt1) that methylates CpG residues and is associated with DNA replication sites during in S phase.…”