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Simplicon® RNA Reprogramming Kit (OKSG)

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.

eCl@ss:
32161000
NACRES:
NA.75
Pricing and availability is not currently available.

application(s)

cell culture | stem cell: suitable

Quality Level

input

sample type induced pluripotent stem cell(s)

shipped in

dry ice

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.

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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.
Research Category
Stem Cell Research
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.

Components

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.

Quality

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.

Storage and Stability

• 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.

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.
Renal Subcapsular Transplantation of PSC-Derived Kidney Organoids Induces Neo-vasculogenesis and Significant Glomerular and Tubular Maturation In Vivo.
van den Berg, CW; Ritsma, L; Avramut, MC; Wiersma, LE; van den Berg, BM; Leuning et al.
Stem Cell Reports null
An integration-free, virus-free rhesus macaque induced pluripotent stem cell line (riPSC90) from embryonic fibroblasts.
Sosa, E; Kim, R; Rojas, EJ; Hosohama, L; Hennebold, JD; Orwig, KE; Clark, AT
Stem Cell Research null
Characterization of induced tissue-specific stem cells from pancreas by a synthetic self-replicative RNA.
Miyagi-Shiohira, C; Nakashima, Y; Kobayashi, N; Saitoh, I; Watanabe, M; Noguchi, H
Scientific Reports null
Articles
Fibroblast growth factors (FGFs) are secreted glycoproteins that regulate several fundamental developmental pathways and help regulate mesoderm and ectoderm patterning in the early embryonic development.
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The Simplicon™ RNA Reprogramming Technology is a next generation reprogramming system that uses a single synthetic, polycistronic self-replicating RNA strand engineered to mimic cellular RNA to generate human iPS cells.
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Human iPSC neural differentiation media and protocols used to generate neural stem cells, neurons and glial cell types.
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Protocols
Stem cell reprogramming protocols to generate human induced pluripotent stem cells (iPSCs) including viral and non-viral RNA based methods.
Read More

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