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.

    New Product of the Week 100410-101010:

pLICO-mWasabi (Promoterless FP Reporter Vector ), listed as product-on-demand, now available, ABP-HL-PE40010 $395.00.

    Promotion of the Week 100410-101010:

Barrier too high to start using virus? Allele lowers it for starters, $500 for bactulo virus protein production, and $300 retrovirus packaging. Code 100310VIVEC, email vivec@allelebiotech.com

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LoxP 4-in-1 iPS Factor on Lentiviral Vectors for Efficient Reprogramming

Putting 4 iPS factors on one lentiviral vector, separated by 2A peptides, has appeared to be more efficient in generating iPS cells than having all 4 factors on individual viruses, at least in a number of cases. Stem cell-like colonies start to appear in about 2 weeks using Allele Biotech’s 4-in-1 lentivirus. In addition to the concerted effects from Oct3/4, Sox2, c-Myc, Klf4, it is also believed that the coordinated silencing of these factors after reprogramming help forming iPS colonies.

The 4-in-1 lentivirus from Allele Biotech contains loxP sites that can be used to remove the 4 cDNAs if so desired. For convenience, a new product kit is offered starting this week to include lenti-nCre in a kit with the 4-in-1 iPS viral products.

New Product of the Week 04-11-10 to 04-18-10: 4-In-One-Vector: Human OSKM Lentiviral Paticles (Oct3/4, Sox2, Klf4 and c-Myc) and Cre Lentiviral Particle kits, Cat # ABP-SC-LVI4IN1C1 or ABP-SC-LVI4IN1C5

Promotion of the Week 04-11-10 to 04-18-10: Single vial 4-in-1 is offered only this week. This product has been well established and validated, one of the reasons smaller packages are not normally offered. As a matter of fact, every batch of the 4-in-1 iPS lentivirus has been sold out.

Update note: Lentivirus inserts into the host chromosome, and is gradually being replaced by footprint-free reprogramming reagents, the best being Allele Biotech’s enhanced mRNA reprogramming factors that feature a patent-pending fusion gene.

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Reprogramming Life

President Obama is expected to lift the ban on federal fund for embryonic stem cell research soon. However, that does not seem to be the hottest topic these days concerning stem cell research. In 2006, Shinya Yamanaka showed that mouse skin cells could be reprogrammed back into something called induced pluripotent stem (iPS) cells by introducing a handful of cDNAs using retroviral vectors. The process was later repeated in human cells and by other groups including those of Thomson and Melton, sometimes with a slightly different set of inducing cDNAs, or with chemicals or shRNA repressing the repressors of the inducer genes.

The iPS cells are not exactly the same as ES cells, and no animals have been created using iPS cells, but they are close enough to be of great interest to lots of people, particularly for basic research purposes. The method to create iPS by reversing chromosomal changes along differentiation pathways appears to be surprisingly simple, like erasing an old audio tape, there may still be acoustic information left if analyzed by the right equipment, but to most people it is as clean as new. You’d wish a few things in life could be reversed that easily!

For labs that are not already in the stem cell field but feel a need to get their feet wet, then they want reagents that are pre-assembled and pre-tested. Such reagents may include: iPS cultures, iPS inducing viral particles, antibodies to stem cell specific markers, cell assays, and even PCR primer sets (synthesizing hundreds of oligos used in the Yamanaka papers alone will take a lot time and unnecessary costs). That’s where a fast-moving, research-oriented company like Allele comes in. We will bring what we think as starter sets for you, and listen to what you think as needed as we along. The new iPS product line will be launched within weeks, hopefully coinciding with our brand new webpages for all our current product lines!

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Monday, February 23rd, 2009 iPSCs and other stem cells No Comments