3 good reasons to select capped polyadenylated mRNA expressing factors for iPSC generation
mRNAs are expression factors that mimic fully processed mRNA. Being the substrate for translation by ribosomes, mRNA expression factors are often preferred over viral vectors for cell reprogramming and iPS cell generation because of the absent risk of integration into the genome. Such RNA-induced pluripotent stem cells (RiPSCs described in 2010 by Warren et al.) are becoming more and more popular. 3 reasons might illustrate RiPSCs’ attractivty.
3 good reasons to select synthetic mRNAs for cell reprogramming and iPSC generation.
Reason #1: Efficient delivery and transient transfection
Delivery to the appropriate compartment in the cell has been a major barrier in gene therapy research.
mRNA expression factors target the cytoplasm, and hence only need to cross the plasma membrane. In contrast, plasmid DNA and most types of viral vectors must reach the nucleus and therefore need to cross an additional membrane barrier. In some cell lines, researchers have observed higher transfection efficiencies with mRNA expression factors compared to DNA plasmids. For some applications transient transfection is desirable. This is much more easily achieved with mRNA expression factors.
Reason # 2: Controlled and fast expression levels
With mRNA expression factors, it is easy to control gene expression.
Expression levels achieved from different promoters used in plasmid DNA or viral vectors can vary dramatically from a cell type to another one. Ectopic promoters are also frequently transcriptionally silenced over time. With integrating viral vectors such as retroviruses or lentiviruses, the number of vector integrations and the location of integration can greatly influence expression in individual cells.
All of these concerns are eliminated with mRNA expression factors because they function post-transcriptionally.
Finally, mRNA expression factors are expressed more rapidly than DNA or virus based approaches since there is no waiting for transcription, splicing, polyadenylation and nuclear export.
Reason #3: Enzymatically synthetized mRNA expressing factors made stable with modified dNTPs
Eliminating the risk of insertional mutagenesis is especially important in applications for phenotype and pathophysiology studies but also regenerative medicine. By example, once iPSCs are made, they are expanded, differentiated and sometimes implanted in animals. In this case, an insertional event can lead to a cancerous phenotype and suboptimal cellular model.
The stem cell community is moving more and more towards non-integrating approaches, such as mRNA expressing factors, cocktails of small bioactive molecules and, optimal control of the cell culture conditions (ex. feeder layers described in a previous post edited by Jean-François Têtu). Repeated administration of a cocktail of synthetic messenger RNAs is also popular. Such in vitro synthetized mRNA expression factors incorporate modified dNTPs to:
- bypass innate cellular antiviral responses,
- boost cell reprogramming kinetics over established viral protocols and,
- fully mimic processed mature mRNAs.
In 2010,Warren et al. were the first to describe a new way to reprogram multiple human cell types by repeated perfusions of mRNAs obtained by in vitro transcription: RNA-induced pluripotent stem cells or RiPSCs. They used 4 to 5 enzymatically synthesized mRNAs (KMOS or KMOSL mRNA cocktails) produced with 5-methyl-CTP (5mC) and pseudo-UTP (psi) from TriLink Biotechnologies. These capped and polyadenylated mRNAs mimic fully processed mature Kfl4, c-myc, Oct4, Sox2 and Lin28 mRNAs.
(1) Warren et al. “Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic ModifiedmRNA” (2010) Cell Stem Cell, Vol. 7 no. 5, 618-630. DOI:10.1016/j.stem.2010.08.012
(2) Zhou et al. Generating optimal pseudouridine and 5-Methylcytidine modified messenger RNA for iPS Cell reprogramming (Poster in collaboration with Beckman Research Inst., Mirus Bio, TriLink BioTechnologies, Baylor College of Medicine)
Want to know more about modified dNTPs to enzymatically produce mRNA for cell reprogramming purposes?
Access to high quality modified dNTPs for enzymatic RNA synthesis has never been so easy. Here, a selection of robust dNTPs that can be used for cell reprogrammning and iPS studies:
- Immune Stimulation Reduction Transcription Nucleotide Set (5-Methylcytidine-5′-Triphosphate, Pseudouridine-5′-Triphosphate, ARCA)
- ARCA (Anti Reverse Cap Analog)
You might also contact experts in cell biology and cell reprogramming!