Mouse and human cells can both be reprogrammed with one cluster of specific miRNAs

The miRNA302/367 cluster was first found to be a direct target for the stem cell-specific factors Oct4 and Sox2, recently Anokye-Danso et al. showed that by overexpressing this miRNA cluster mouse and human cells can be reprogrammed without the OSKM factors. Moreover, according to the publication in Cell Stem Cell, miRNA-mediated reprogramming is “up to two orders of magnitude” more efficient than OSKM overexpression (but the authors used individual Oct4, Sox2, Klf4, and c-Myc lentiviruses, instead of a polycistronic virus such as Allele’s lenti-iPS-4-in-1).

To reprogram mouse embryonic fibroblasts (MEFs), suppression of chromatin remodeling factor Hdac2 is necessary when using miRNA for iPSC isolation. Surprisingly, the Hdac2 level is low in human fibroblasts, which do not need an Hdac inhibitor such as valproic acid (VPA) for reprogramming. Oct4-GFP positive cells (stem cells) are observed only 7 days post infecting MEFs with the miRNA302/367, and hundreds colonies appear per 10 thousand cells. When using human fibroblasts, iPSCs form at 18 to 26 days, at an efficiency of approximately 10%, which is significantly higher than using individual OSKM viruses.

The high efficiency from using miRNA for reprogramming is likely due to the fact that miRNAs can target hundreds of mRNAs, compared to providing one mRNA at a time. Although this study concluded that the miRNA302/367 expressing lentivirus was eventually silenced post stem cell induction, emphasis must still be placed on finding a non-integrating method to deliver this miRNA cluster.

New Product of the Week: Chemically synthesized miRNAs by your own design, email oligo@allelebiotech.com for details.

Promotion of the week: Promotion of the week: save 10% on AlleleBalanced Luciferase Assay Kits. Email the code Luc10 to abbashussain@allelebiotech.com to redeem this offer.

Tags: Anokye-Danso, Hdac2, human fibroblasts, iPSC, lentivirus, MEFs, miRNA derived iPSC, miRNA lentivirus, miRNA-mediated reprogramming, OSKM, VPA

Working on the Wnt signaling pathway

Wnt proteins form a family of highly conserved, secreted glycolipoproteins that regulate cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis. Mutations in Wnt genes or the Wnt signaling pathway components are often linked to human birth defects, cancer and other diseases.

Since the discovery of the Wnt-1 gene 27 years ago, a complex Wnt signaling network with many components having multiple distinct roles and acting in different cellular compartments has been established. The studies of Wnt signaling in human diseases, in stem cell biology and tissue regeneration holds promise for translational medicine.

A few commonly asked questions regarding studies on the Wnt pathways will be touched upon in this blog series:

How to activate the Wnt Signaling Pathway?

1) Recombinant Wnt Proteins: there are several commercial sources for recombinant Wnt proteins. As most of them are produced in bacteria, endotoxin, purity and active concentration are of primary concern. From our own experience using commonly used commercial Wnt proteins, the activities vary from batch-to-batch, and sometimes the products are simply inadequate.

2) Conditioned Medium: this approach is convenient and of low cost. ATCC provides two mouse fibroblast cell lines, which over-express mouse Wnt3A (CRL-2647) and mouse Wnt5A (CRL-2814). Researchers can maintain the cell lines and collect conditioned medium whenever they need Wnt proteins. However, these are the only two Wnt over-expressing cell lines available on the market, yet there are 19 members in the Wnt gene family. If you are planning on working with Wnt proteins other than 3A and 5A, you would have to develop relevant cell lines on your own. On the other hand, because over-expression of Wnt proteins will also activate the secretion of some growth factors, such as FGFs, the conditioned medium should be a mixture of several kinds of secreted factors.

3) Over-expression Wnt proteins directly in your cells of interests: Over-expression is a basic strategy for functional biology research. One of the barriers for introducing cDNAs into cells is the transfection efficiency. The advent of retroviral transfection (MMLV based or Lenti based) technically resolves this problem, as the transduction efficiency can reach almost 100%. However, highly efficient retrovirus packaging remains a difficult process for most research labs.

Allele Biotech plans to introduce all the human Wnt cDNAs on its HiTiter™ Expression Lentivirus platform as shelf products. The package size will be 5 vials, each vial 100 µl, the titer is 10e8 TU/ml at a price of $799. This product line will be under “Product-on-Demand”, with clones ready but packaged only upon order for the first time. First-time orders will qualify for a 10% discount, if the first-time customer provides the cDNA, there will be an additional 20% discount. These lentiviruses can be used directly in target cells or to build over-expressing cell lines for conditioned medium. Next topic is How to inhibit Wnt signaling pathway?

New Product of the Week 120610-121310: Human miRNA minigene on lentivirus with RFP reporter, ABP-RP-MI221LP

Promotion of the Wee 120610-121310: 5% off virus packaging if you use our FaceBook code “ViralPackaging” at shop.allelebiotech.com (beta-test new web).

Tags: cell fate, cell polarity, cell proliferation, conditioned medium, growth factors, lentivirus, signaling pathways, Wnt

How to order virus packaging service

Step 1: Please review the following before ordering Allele’s retrovirus packaging service.

Verify that your plasmids for virus production meet the following criteria:

No internal polyA signal or polyA signal immediately downstream of the coding cistron.

Avoid toxic genes, unusual secondary structures, and expression cassettes > 8kb (5’ to 3’ LTR).

If these concerns can not be satisfied in your vector design, please contact our technical specialist (vivec@allelebiotech.com) for further discussion, as Allele’s proprietary packaging technology might be able to help.

Step 2: Select the service:

Allele offers multiple project discounts: a 5% discount for ordering 3-5 packaging services, and a 10% for 6-10 packaging services.

Additional information can be found here.

Step 3: Send us 10 ug of endotoxin-free plasmid (blue ice shipping) for virus packaging.

To facilitate the completion of the custom virus packaging projects, Allele offers a variety of retro/lentivirus plasmids as well as viral plasmid subcloning and endotoxin-free plasmid preparation services. Please contact us (oligo@allelebiotech.com or 858-587-6645) for further details.

Once your order is placed, we will contact you about plasmid shipment and provide you with a time frame for completing the project.

Note: If the total project price is more than $1000, a non-refundable down payment (30% of the total service price) is registered before the project is initiated.

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Promotion of the Wee 120610-121310:

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Tags: custom virus packaging, EF1a, lentivirus, lentivirus packaging, LTR, retrovirus, retrovirus titering, SV40

Features of Allele’s Virus Packaging Services

High Titer
Allele Biotech’s HiTiter™ Viral Packaging Services, using proprietary technologies that are unique and highly efficient, can easily yield 10e8 to 10e9 TU/ml lentivirus/retrovirus without concentrating steps. With a concentration step 10e9 to 10e10 TU/ml can be achieved, which is ideal for in vivo research. It’s the world’s most powerful viral packaging platform.

Quick Turnaround
Allele Biotech has consolidated its procedure of HiTiter™ Viral Packaging Services as a standard production-line resulting in a quick turnaround. Typically, a standard 10e8 TU/ml 2ml packaging service is completed in one week and delivered in less than two weeks. You send us virus transfer plasmids, wait a week or two, and receive high titer ready-to-use virus. Imagine how our ingenuity and innovation can pave the way for your research!

Ideal for Complicated Constructs
Viral packaging, especially for complicated constructs, has been intrinsically difficult for most researchers. Now with Allele Biotech HiTiter™ Viral Packaging Services, you have an ideal option for packaging even the most demanding constructs! We have successfully developed the 4-In-1 iPSC Generation Lentivirus, expressing 4 genes (hOct3/4, hSox2, hKlf4 and hc-Myc) in one lentiviral construct, which has been very popular in the filed of stem cell research.

Fluorescent Proteins & Drug Resistance
Allele Biotech and our collaborators design, evolve, and select new FPs that suit different applications. These brighter fluorescent proteins, sometimes together with drug resistance genes, have been incorporated into our lentiviral/retroviral systems. When you order our viral packaging service, you will automatically receive a 20% discount for all our listed viral plasmids.

Full RNA Interference Services
We have RNAi lentiviral packaging services, RNAi validation services, and RNAi screening services, which can meet different demands in the filed of RNA Interference. The RNAi lentiviral packaging services include Gene-To-Silence™, Sequence-to-Virus™ and DNA-to-Virus™. The plasmids we use for these services have build-in FPs and drug resistance genes, convenient for virtually all research purposes.

New Product of the Week 111510-112110:

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Promotion of the Wee 111510-112110:

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Tags: custom viral packaging, high titer, lenti-cre, lentivirus, packaging lentivirus, pre-packaged viruses, retrovirus, RNAi screening

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

Tags: c-myc, in vitro transcription, integration-free, iPSC, iPSCs, Klf, lentivirus, modified synthetic mRNA, mRNA iPSCs, mRNA transfection, Oct, retrovirus, RiPSC, RiPSCs, Sox