Pluripotency Induction: Episome basedGenerate Transgene-free iPSCs
• Plasmid mixture with Oct4, Sox2, Klf4, L-Myc, Lin28, shRNA-p53,
miR302/367 and GFP
• Simple protocol for reliable reprogramming
• Reprogram elderly fibroblast cells efficiently
• Compatible with feeder or feeder-free conditions
Generate Transgene-free iPS Cell Lines Rapidly
The use of four reprogramming factors delivered via retroviral transduction of cells was initially established as a powerful way to reprogram any somatic cell into an ES-like state. These reprogrammed cells are capable of differentiating into any cells representing the three germlines, presenting a system that mirrors human ES cells. While such systems are quite useful for in vitro studies of differentiation, the challenge of adapting these virally-derived iPSCs for in vivo applications (e.g. cell therapy) is quite high, owing to the potential for random integration and subsequent risk of mutagenesis from viral-mediated delivery of reprogramming factors.
SBI has developed and validated a system that uses a non-viral, non-integrating, plasmid-based reprogramming technology and is a unique alternative to traditional retroviral-based reprogramming of cells. SBI´s Episomal iPSC Reprogramming System (catalog #SC900A-1) is based on the Epstein-Barr Nuclear Antigen-1 (oriP/EBNA-1) that has been proven to generate iPSCs very efficiently without the risk of transgenic sequences inserted into the target cell genome.
How does the oriP/EBNA-1 system work?
Unlike traditional plasmid systems, the oriP/EBNA-1 system replicates in synchrony with the host genome by anchoring itself to the host chromatin and replicating during cell cycle divisions. The episomal plasmids are naturally lost at up to 5% per cell division cycle. SBI´s episomal reprogramming system contains Oct4, Sox2, Klf4, L-myc, Lin28, shRNA-p53, and miR302/367 cluster reprogramming factors, along with a GFP marker for monitoring transfection efficiency and plasmid loss over time. These transgene-free iPSCs have the capability to be utilized for a broad range of applications, including pre-clinical research and human cell therapy research, thus further delivering on the promise of iPS cells.
Generate Transgene-free iPS Cell Lines Rapidly
Transfect target cells only once with the Episomal Reprogramming plasmid mixture (Catalog# SC900A-1-SBI). Transfection efficiency can be monitored using the co-expressed GFP marker. Visible iPSC colony formation can be observed in as little as two weeks and is complete in 25 days with 70% more iPSC colonies generated compared to standard OSKM methods. The data below in the left panel show the GFP signals in Human adult primary dermal fibroblasts one day after electroporation. The progression and derivation of validated iPS cells under feeder-free conditions with SBI´s PSGro® Human ESC/iPSC Growth Medium for feeder-free conditions (Catalog# SC500M-1-SBI) plus SBI´s PSGen® Reprogramming Supplement (Catalog# SC551M-1-SBI) can be viewed in the right hand panels.
The Episomal oriP/EBNA-1 reprogramming system also works on difficult-to-reprogram source cells
Older patient dermal fibroblast samples can often be difficult to reprogram. The Episomal oriP/EBNA-1 Reprogramming plasmid mix also works on these difficult-to-reprogram source cells. The panel to the left shows successful iPSC generation using a passage 8 human dermal fibroblasts from a 60 year-old patient sample, whereas standard Yamanaka OSKM factors (right panel) failed to induce any iPSC formation.
Robust and Healthy iPSCs Generated
The iPSC colonies generated using SC900A-1-SBI have clear morphology of reprogrammed cells and express stem cell-specific markers. The data below depict the characterization of SC900A-1 iPS cell colonies by Alkaline phosphatase (AP) staining and immuno-staining of pluripotency markers, Nanog, Oct4, Sox2, SSEA3, TRA-1-60 and TRA-1-81 (Using SBI´s complete Antibody and AP staining Kit, catalog# SAB-KIT-1-SBI).
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007 Nov 30;131(5):861-72.
Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62.
Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 2009 May 8;324(5928):797-801.
|Episomal iPSC Reprogramming System (EBNA-1 based)||SC900A-1-SBI||5 reactions||986 €||DETAILS||Add to Cart|