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Human OKSG-cMyc TagRFP Simplicon RNA

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eCl@ss:
32161000

description

EMD Millipore’s Simplicon® OKSG-cMycTagRFP RNA 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, Glis1 and c-Myc along with a red fluorescent protein (TagRFP).

Quality Level

technique(s)

nucleic acid detection: suitable (RNA)

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, (3) daily transfections of cells using four synthetic mRNAs over a 14 day period (i.e. mRNA based)2, and (4) the inability to directly assess transfection or viral transduction efficiency of the introduced reprogramming factors.

Our Simplicon OKSG-cMyc TagRFP RNA uses a safe and efficient method to generate integration- and virus-free human iPSCs using a single transfection step. The technology utilizes a single self-replicating Venezuelian equine encephalitis (VEE) RNA species that expresses the reprogramming factors (RF) ORFs3 (OKSG-cMyc; Oct4, Klf4, Sox2, Glis1 and cMyc) along with a red fluorescent protein (TagRFP). The Simplicon RNA replicon is a synthetic polycistronic VEE-RF RNA that is capable of self-replicating in a limited number of cell divisions. The OKSG-cMyc transgenes are especially useful for iPSCs generation from somatic cells that are more difficult to reprogram (i.e. slower proliferating cells or aged somatic cells) while the TagRFP provides a rapid assessment of transfection efficiency. Presence of the TagRFP transgene also allows for optimization of the transfection conditions in hard- to- transfect somatic or primary cells.

Application

Advantages of the Simplicon OKSG-cMyc TagRFP RNA:

• Integration-free, footprint-free iPSCs generation. No risk of genomic integration.
• Safe, virus-free, synthetic polycistronic RNA replicon (all five reprogramming factors in a single RNA strand)
• TagRFP reporter gene construct embedded in the RNA replicon that allows visualization and quantification of the transfection efficiency of the Simplicon RNA.
• Only one single transfection required. The RNA replicon is able to self-replicate, eliminating the need for additional daily transfections of multiple individual mRNAs over a 14-day period.
• Efficient and rapid reprogramming.
• No screening required to ensure the absence of viral remnants.
• Controlled elimination of synthetic VEE RNA replicon by the removal of B18R protein.
• Validated for reprogramming in feeder-free and feeder-based culture conditions.
• The OKSG-cMyc transgenes are especially useful for iPSCs generation from somatic cells that are more difficult to reprogram (i.e. slower proliferating cells or aged somatic cells) while the TagRFP provides a rapid assessment of transfection efficiency. Presence of the TagRFP transgene also allows for optimization of the transfection conditions in hard- to- transfect somatic or primary cells.

Other Notes

Concentration: Please refer to lot specific datasheet.

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.

Storage Class

12 - Non Combustible Liquids

wgk_germany

WGK 2

flash_point_f

Not applicable

flash_point_c

Not applicable


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