Description
General description
Various methods utilizing viruses, DNA, RNA, miRNA and protein have been developed to generate integration-free induced pluripotent stem cells (iPSCs). Disadvantages to existing methods include: (1) low reprogramming efficiency (i.e. DNA and protein), (2) a lengthy requirement for negative selection and subcloing steps to remove persistent traces of the virus (i.e. Sendai virus)1 and (3) for daily transfections of four individual in vitro generated mRNAs over a 14 day period (i.e. mRNA based)2.
EMD Millipore’s Simplicon RNA Reprogramming Kit is a safe and efficient method to generate integration free, virus-free human iPS cell using a single transfection step. The technology is based upon a positive strand, single-stranded RNA species derived from non-infectious (non-packaging), self-replicating Venezuelian equine encephalitis (VEE) virus3. The Simplicon RNA replicon is a synthetic in vitro transcribed RNA expressing all four reprogramming factors (OKS-iG; Oct4, Klf4, Sox2 and Glis1) in a polycistronic transcript that is able to self-replicate for a limited number of cell divisions.
Application
Advantages of the Simplicon RNA Reprogramming Kit:
• Integration-free, footprint-free iPS cells. No risk of genomic integration
• Safe, virus free, synthetic polycistronic RNA replicon (all four reprogramming factors in 1 RNA strand)
•Only 1-day transfection required. The RNA replicon is able to self-replicate, elliminating the need for laborious daily transfection of multiple individual mRNAs over a 14 day period.
• Efficient and rapid reprogramming.
• No screening required to ensure viral remnants are not present.
• Controlled elimination of synthetic RNA replicon by removal of B18R protein.
• Validated for reprogramming in feeder-based and feeder-free culture conditions.
EMD Millipore’s Simplicon RNA Reprogramming Kit is a safe and efficient method to generate integration free, virus-free human iPS cell using a single transfection step of self-replicating synthetic RNA for Oct4, Klf4, Sox2 and Glis1.
Research Category
Stem Cell Research
Preparation Note
• VEE-OKS-iG and B18R RNAs: Stable for 4 months from date of receipt when stored appropriately at -80°C. For best recovery, quick-spin the vial prior to opening. Thaw on ice. While on ice, aliquot into sterile, nuclease-free eppendorf tubes and store at -80°C. Limit repeated freeze-thaw cycles. Use in a sterile RNAse-free environment.
• Human recombinant B18R protein: Stable for 4 months from date of receipt when stored appropriately at -80°C. For best recovery, quick-spin the vial prior to opening. Thaw on ice. While on ice, aliquot B18R protein into sterile, nuclease-free, low protein-binding eppendorf tubes and store at -80°C. B18R protein must be kept on ice in order to avoid degradation. Limit repeated freeze-thaw cycles. Use in a sterile RNAse-free environment.
• Human iPS Reprogramming Boost Supplement II: Stable for 4 months at -20˚C from date of receipt. Upon first thaw, aliquot into smaller working volumes and freeze at -20°C. Upon addition of the small molecule components to the media, filter the supplemented media with a 0.22 μM filtration unit and stored at 2-8˚C. For optimal results, prepare sufficient supplemented media for a 1 week supply of media changes.
Analysis Note
Tested to confirm the generation of iPS cells from p6 human foreskin fibroblasts. Other cell types have not been tested and thus similar results can not be guaranteed.
Other Notes
1. VEE-OKS-iG RNA: (Part No. CS210583) One (1) vial containing 10 µL of RNA (1 ug/uL). Store at -80°C.
2. B18R RNA: (Part No. CS210584) One (1) vial containing 10 µL of RNA (1 ug/uL). Store at -80°C.
3. Human Recombinant B18R Protein, Carrier-Free: (Part No. GF156) One (1) vial containing 50 ug of 0.5 mg/mL stock of B18R protein. Store at -80°C.
4. Human iPS Reprogramming Boost Supplement II: (Part No. SCM094). Store at -20°C.
Legal Information
SIMPLICON is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
