In vitro transcription templates for large-scale synthesis of mRNA encoding six iPS reprogramming factors.

iPS Induction IVT Templates,

n 2010, Warren et al. illustrated a footprint free method to reprogram adult cell to iPSCs using modified mRNA molecules. This method was groundbreaking for two reasons, the use of modified RNA bases mitigated cell immune response to transfected mRNA, and the efficiency of induction was higher than viral transfection. Through the use of exogenous gene expression, iPSCs can be produced without genome rearrangement, removing one of the greatest barriers to iPSC-based therapy.

Box 1 | Product List

iPS Induction System Content
Description iPS induction IVT Template Set (OSKMNL and mWasabi Oct3/4 IVT Template M3O IVT Template SOX2 IVT Template c-Myc IVT Template Klf4 IVT Template Nanog IVT Template Lin28 IVT Template Cat. No. ABP-SC-SEIPSSET ABP-SC-SEOCT34 ABP-SC-SEM3O ABP-SC-SESOX2 ABP-SC-SECMYC ABP-SC-SEKLF4 ABP-SC-SENANOG ABP-SC-SELIN28 Qty
5 g x 7 5 g 10 g 5 g 5 g 5 g 5 g 5 g

Description
The iPS Induction IVT templates gives the ability to perform footprint-free iPSC induction using mRNA transfection, a new reprogramming technology thats faster and more efficient than conventional virus-based approaches. Allele offers in vitro transcription templates for large-scale synthesis of mRNA encoding the six human reprogramming factors (Klf4, Myc, Oct4, M3O, Sox2, Lin28, and Nanog), fluorescent reporters (mWasabi, mTFP1, and LanYFP), Luciferase, and Cre Recombinase. These linear DNA templates are ready to use directly in IVT reactions. Within each template, the coding sequence for a protein is flanked by 5 and 3 untranslated regions designed to maximize the translational efficiency and cytoplasmic stability of the mRNA and validated in the iPSC application.

Fluorescent Controls
mWasabi IVT Template mTFP1 IVT Template LanYFP IVT Template ABP-FP-SEMWASABI Coming Soon Coming Soon
5 g 5 g 5 g

Functional Proteins
Cre IVT Template Luciferase IVT Template ABP-RP-SECRE ABP-RP-SELUC
10 g 10 g

The templates drive incorporation of 120-nt polyA tails into RNA transcripts, eliminating the need to enzymatically polyadenylate IVT products before use. Enough template material is provided to make hundreds of micrograms of individual mRNAs. With this abundance of mRNA you can have the freedom to explore different factor cocktails and optimize reprogramming conditions for your cell type of interest.

Features
Templates contain the T7 phage polymerase promoter allowing the widely available T7 polymerase to be used. Inherently non-mutagenic IVT template for footprint-free iPSC production Eliminates safety concerns and biocontainment issues associated with virus Potential to Reprogram human fibroblasts with colonies in as little as 16 days One kit can be used to make over two milligrams of synthetic mRNA

Figure 1 | iPS Induction using mRNA

Bright-field images taken during the derivation of RNA-iPSCs (RiPS) from dH1f fibroblasts showing early epitheloid morphology (day 6), small hESC-like colonies (day 17), and appearance of mature iPSC clones after mechanical picking and expansion. Warren et al., Cell Stem Cell (2010)

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Overview

n order to efficiently and effectively reprogram cells into iPSC using mRNA as a method of gene delivery, multiple steps must be taken to ensure that:

mRNA is successfully invitro-transcribed with proper modifications in effort to suppress target cell immune activity towards mRNA molecules. mRNA is of suitable purity mRNA products are delivered into target cells

Allele Biotech conveniently offers imperative components to produce footprint free iPSC as described in Warren et al.2010 publication. The following table outlines necessary components required, in addition to the iPS IVT templates, for cell reprogramming.

Box 2 | Stealth Express Overview

Component 2.5x Nucleotide Mix w/ Cap Analog

Cat. No. ABP-PP-NTPMIX

Description Blend of components needed for IVT reactions. Included in the Ready-Mix cocktail:
Modified UTP (Pseudouridine) Modified CTP (5-Methylcytidine) ATP GTP ARCA (Anti-Reverse Cap Analog)

Modified bases are intended to ameliorate immunological responses towards the mRNA product.

SurfaceBind RNA Purification Kit Transfection Reagent B18 Stem Cell Media

ABP-PP-SBRNA025 Coming Soon Coming Soon Coming Soon

For purification of RNA products Cationic vehicle to facilitate endocytic delivery of mRNA. Immune inhibitor allows for repeat mRNA transfections. Cell media optimized for the culturing of ES and iPS cells.

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