Strategies for simultaneous and successive delivery of RNA
- Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the secondAdvanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.…
Author details: | Hanieh MoradianORCiD, Andreas LendleinORCiDGND, Manfred GossenORCiDGND |
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DOI: | https://doi.org/10.1007/s00109-020-01956-1 |
ISSN: | 0946-2716 |
ISSN: | 1432-1440 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/33146744 |
Title of parent work (English): | Journal of molecular medicine |
Publisher: | Springer |
Place of publishing: | Heidelberg |
Publication type: | Article |
Language: | English |
Date of first publication: | 2020/11/04 |
Publication year: | 2020 |
Release date: | 2023/01/25 |
Tag: | co-expression; in vitro synthesized mRNA; integrated co-transfection; parallel co-transfection; successive; transfection; transfection methods |
Volume: | 98 |
Issue: | 12 |
Number of pages: | 13 |
First page: | 1767 |
Last Page: | 1779 |
Funding institution: | Helmholtz AssociationHelmholtz Association; Helmholtz Graduate School of; Macromolecular Bioscience (MacroBio) [VH-GS-503]; German Federal; Ministry of Education and Research through the Program Health Research; [13GW0098]; Projekt DEAL |
Organizational units: | Wirtschafts- und Sozialwissenschaftliche Fakultät / Wirtschaftswissenschaften |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer review: | Referiert |
Publishing method: | Open Access / Hybrid Open-Access |
License (German): | CC-BY - Namensnennung 4.0 International |