Making Transfection-Grade mRNA by IVT (In Vitro Transcription)

RNases are an often feared in molecular biology labs because of their high stability and ominous presence in virtually all living systems. Consequently, people who work with RNA are trained to exercise extreme caution to avoid RNA degradation: change gloves often because human hands ooze RNases; use only sterilized labware as microbes may be sources of RNases; for surfaces that can’t be autoclaved, use sprays like “RNase Zap” (SDS- or guanidine-containing solutions). Such cautionary steps are especially necessary when dealing with low abundance RNA samples.

RNAs can be produced by in vitro transcription (IVT), a simple reaction requiring only a DNA template (double-stranded or even single-stranded DNA as long as the promoter region is double-stranded), RNA polymerase (from T7, SP6, or T3 phage), NTPs, and a reaction buffer that provides appropriate salt and pH. Standard NTPs may be replaced with modified ones to either increase stability or to reduce immune-response when transfected into cultured cells. Additionally, a 5’ cap structure may be added during IVT for further stabilizing mRNAs inside the cells post transfection. Using a commercially assembled kit, one can routinely produce 40-50 µg of mRNA from 1 µg of DNA template in a single 20-50 µl reaction.

At such high concentrations, IVT mRNAs are not nearly as sensitive to RNase-mediated degradation as low-abundance samples. The mRNA can be easily observed on agarose gels that are regularly used for DNA, and their integrity can be monitored after transcription or storage. In most cases one distinct band of mRNA from an IVT reaction is obtained as long as a clean DNA template is used. Preparing a good, uniform IVT template is critical to prevent aberrant products. By using high quality templates, IVT mRNA produced in your own lab are often higher in quality than mRNAs purchased from current commercial sources (Figure in Blog shows mRNAs generated by IVT for R-iPSC). Sometimes there are minor bands created during IVT, but they normally do not interfere with the intended uses of the mRNA, and can be purified away with a purification kit (by using a discriminating purification scheme such as Allele Biotech’s Surface Bind RNA Purification, smaller species can be specifically removed, a separate topic for another blog).

Once produced, mRNAs can be stored at -20C for months, or -80C nearly indefinitely.

IVT mRNA for iPSC generation

mRNAs generated by IVT for R-iPSC

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Methods of iPSC Generation Update

Induced pluripotent stem cells can be directly generated from adult cell cultures through the introduction of a group of factors, e.g. Oct4, Sox2, Klf4, and c-Myc (the Yamanaka factors) [Takahashi and Yamanaka, 2006]. Additional factors such as Nanog and Lin28 can either substitute some of the Yamanaka factors or supplement them for higher reprogramming efficiency [Yu et al. 2007].

The original pluripotent stem cells induction methods involved retrovirus or lentivirus that would leave foot-print in the host genome, a concern for clinical use of iPSCs. Several groups have tried to create iPSCs without integrating viruses, such as using small molecules, directly delivering proteins instead of cDNAs, viruses with RNA genomes, episomal systems, or removable elements such as PiggyBac or Sleeping Beauty transposons. From the literature and our first-hand experience in the iPS market, none of these methods has become a widely applicable tool, mostly due to impractically low reprogramming efficiency.

In addition to low efficiency, RNA viruses, such as the sendai virus, are still viruses and have virus-associated risks. Episomal plasmids or removable transposons still involve DNA, so the possibility of genomic integration by recombination remains. In case of some transposons such as PiggyBac, there is an additional question about the degree of removal – whether it is certain that all integrated transposons, often inserted within genes, are deleted; in case of transposons similar to Sleeping Beauty, the small footprints they leave behind may post a concern.

The method of choice for generating zero-footprint iPSCs should clearly be RNA-based without the involvement of virus. Luigi Warren and his former colleagues at Harvard demonstrated that by using in vitro transcribed iPS factor mRNAs with modified CTP and UTP, and 5’-cap can effectively reprogram a number of different human as well as mouse cells. The efficiency even exceeds those by using retrovirus or lentivirus by 10 to 100 fold. Furthermore, the RiPSCs created with mRNAs appear to be closer to hESCs as shown by expression profiling.

Very recently, a few miRNAs that have high expression levels in stem cells were shown to be able to reprogram mouse and human somatic cells when expressed together from a lentivirus [Anokye-Danso et al. 2011]. while that work used lentivirus, thus not directly applicable to the current project, Miyoshi et al. later showed that by using synthesized mature miRNA (overlapping but not the same set of miRNAs as used by Anokye-Danso et al.) reprogramming cold be achieved without viral infection. We believe that this is a promising method and would like to pursue it further and to find out whether these mi-iPSCs relate to hESCs as closely as R-iPSCs. Because transfecting synthetic miRNAs “does operate at considerably lower efficiency” in terms of iPSC creation [Miyoshi et al. 2011],alternative protocols may include transfecting the iPS factor mRNAs together with various miRNAs at different doses and frequencies.

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Blog References: Warren, L. et al. “Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA” 2010 Cell Stem Cell 7(5): 618-30

Anokye-Danso, F. et al. “Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency” 2011 Cell Stem Cell 8(4): 376-88

Miyoshi, N. et al. “Reprogramming of mouse and human cells to pluripotency using mature microRNAs” 2011 Cell Stem Cell 8(6): 633-8

Kim, H. et al. “miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells” 2011 Cell Stem Cell 8(6): 695-706

Takahashi, K. and Yamanaka, S. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors” 2006 Cell 126(4): 663-76

Yu, J. et al. “Induced pluripotent stem cell lines derived from human somatic cells” 2007 Science 318(5858): 1917-20

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Wednesday, August 31st, 2011 iPSCs and other stem cells No Comments

Expression of iPS Factors from Transfected mRNA

Differentiated cells can be reprogrammed to pluripotency by enforced expression of certain combinations of stem cell-specific protein factors in them. The power of this method was first demonstrated by Yamanaka’s group using retroviruses carrying Oct3/4, Sox2, c-Myc, and Klf4. Alternative factors such as Lin28 and Nanog, and additional factors such as the human telomerase gene hTert and shRNA against p53 were also shown to contribute to reprogramming. From the very beginning it was realized that viral integration would pose a major problem in using the induced pluripotent stem cells (iPSCs) for clinical purposes. There have been multiple attempts to circumvent this problem by using non-integrating vectors such as plasmid, minicircle DNA, adenovirus, baculovirus, removable transposons, episomal DNA, or by introducing recombinant proteins with a transmembrane domain into target cells. From reports in the field and customer feedbacks it seems that retroviral or lentiviral systems are still the most efficient in reprogramming. mRNA is about the only option left unreported, until an article by Warren et al was published in Cell Stem Cell online recently.

From that report, it is clear that the reason that it took so long for RNA-induced iPSCs (RiPSCs) to appear in the literature was because synthetic mRNAs activate interferon responses in mammalian cells, reminding us of the early days of RNAi. The authors took a number of steps to reduce interferon responses, including adding a 5’-cap (actually a fairly standard step in in vitro transcription), using a phosphatase to remove 5’ triphosphates on uncapped mRNAs, and using modified C and U bases (5-methucytidine or 5mC and pseudouridine or psi) during T7 promoter-driven in vitro transcription. The prepared mRNA was then administered everyday for 17 days at an amount not clearly defined in the paper. The main benefit of this method is of course that there is no gene integration to alter the chromosome. The efficiency of the new method was also compared to using viral vectors and it was shown that 1.4% conversion efficiency was achieved vs retroviral systems’ 0.01% (although we have experienced better results using lentivirus, at least the 4-in-1 version).

The DNA templates used for in vitro transcription of the iPS factors were created by multiple PCR reactions and bridged ligation; it could also be done by other cloning strategies. For those excited about trying this new way of making iPSCs, the major hassle would be preparing modified mRNAs good and abundant enough for 17 consecutive transfections. Allele Biotech would like to provide custom services, before offering shelf products, for creating such mRNAs as the method sounds potentially very helpful to many researchers in the iPSC field.

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