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Cell Fate Regulating Enzymes

iCP-RFs

“improved Cell-Permeable Reprogramming Factors”

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Description

Safe & Highly Efficient Cell-Penetrating Peptide-Based Nuclear Reprogramming Kit
The improved cell-permeable reprogramming factors (iCP-RFs) kit contains cell-permeable protein-based reagents necessary for nuclear reprogramming. Thanks to Cellivery’s novel Therapeuticmolecule Systemic Delivery Technology  platform the iCP-RFs are capable of direct penetration of the cell membrane, not requiring virus-mediated delivery, to gain access to the intracellular compartments. Due to the delivery mechanism, iCP-RFs are free from epigenetic safety problems accompanied by conventional methods (Masato Nakagawa et al. Nature Biotechnology 2008), like insertional mutagenesis. Moreover, iCP-RFs can achieve more efficient reprogramming at lower concentrations (0.1~0.5 μM) and accelerate the emergence of iPSC colonies by 10~100 times compared to the existing viral vector-based methods (Trond Aasen et al. Nature Biotechnology 2008). 



Necessity of Generating Protein-Derived Induced Pluripotent Stem Cells (iPSCs) 
Reprogramming factors (RFs) promote the conversion of somatic cells into cells resembling embryonic stem cells called induced pluripotent stem cells (iPSCs). RFs known to enhance self-renewal and inhibit differentiation, include Octamer-binding Transcription Factor 4 (OCT4), Sex Determining Region Y-box2 (SOX2), Kruppel-like Factor 4(KLF4), V-myc Myelocytomatosis Viral Oncogene Homolog (CMYC), Nanog Homeobox (NANOG), and Lin-28 Homolog (LIN28). In order to solve the problem of conventional gene transfer using viruses, there has been a great demand for a technique of directly transferring the RF into a cell as a protein. 

Protein-based approaches avoid problems associated with gene delivery, notably mutagenic effects of inserted gene sequences (Dohoon Kim et al. Cell Stem Cell 2009) and variable expression of RF proteins from integrated expression vectors. Protein-based delivery methods allow the amount and duration of RF proteins to be titrated exactly for optimal cellular reprogramming. 

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Table 1. Various methods for generation of iPSCs and their limitations


Protein-based approaches avoid problems associated with gene delivery, notably mutagenic effects of inserted gene sequences (Dohoon Kim et al. Cell Stem Cell 2009) and variable expression of RF proteins from integrated expression vectors. Protein-based delivery methods allow the amount and duration of RF proteins to be titrated exactly for optimal cellular reprogramming.

Properties
  • Cat. No : CV-10
  • Expression Host : E. coli
  • Concentration : 
  • Purity : 
  • Endotoxin : 
  • Specific Activity :
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FAQ / Q&A

1. Why is the daily treatment for 6 hours?

The treatment duration was experimentally determined condition which expresses maximum biological activity

2. How long does it take to obtain iPSCs?

Early emergence of colonies can be observed in 5 days

3. Can other additives be added?

Adding Leukemia Inhibitory Factor (LIF) or Vitamin C can increase the reprogramming efficiencies.

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Reference
Cautions

· Always store in deep freezer (under -80℃). · Always thaw iCP-RFs on ice before use and keep it over the ice during the experiments. · Avoid freeze/thaw cycle · (It is highly recommended that the iCP-RFs be aliquoted for an one experiment operative dose each and stored in the fridge).


Ⅰ. Preparation of iCP-RF Cocktail


To prepare iCP-RF Cocktail add iCP-OCT4, iCP-CMYC, iCP-NANOG, iCP-LIN28, iCP-KLF4 and iCP-SOX2 to serum free DMEM (0.1 μM iCP-KLF4 and 1 μM for remaining iCP-RFs).


Ⅱ. Cell Culture and iCP-RFs Treatment


To prepare iCP-RF Cocktail add iCP-OCT4, iCP-CMYC, iCP-NANOG, iCP-LIN28, iCP-KLF4 and iCP-SOX2 to serum free DMEM (0.1 μM iCP-KLF4 and 1 μM for remaining iCP-RFs).
1. Human somatic cells are cultured until they are 75-90% confluent in a single well of 6-well plate
2. Once the desired confluency is reached, add iCP-RF cocktail gently into the wells, 
3. After the treatment of iCP-RFs, incubate the plate at 37℃ for 6 hours.
4. Remove the supernatant and wash the cells 2~3 times with pre-warmed PBS or serum free DMEM. 
5. Subsequently, add pre-warmed growth medium into the well and incubate the plate at 37℃ overnight.
6. Conduct the daily treatment of iCP-RFs and washing steps daily until iPSC-like colonies are formed.


Ⅲ. Feed and picking the cells


1. Prepare complete human iPSC medium.
2. When formation of colonies is observed, terminate the iCP-RFs treatment and replace growth medium with 2ml of human iPSC medium.
3. Every 24 hours, exchange 70-100% of the medium to a fresh iPSC medium.
4. Observe the growth of colonies to an appropriate size for transfer. 
5. The day before transferring the colonies prepare MEF (mouse embryonic fibroblast) culture plates or feeder-free protein coated (ex. Vitronectin) in12-well plates. 
6. When Colonies are ready for transfer, conduct live staining by using Alkaline Phosphatase (AP) or TRA-1-60 for picking reprogrammed colonies.
7. Pick AP or TRA-1-60 positive colonies and transfer them onto prepared MEF culture plates or feeder-free protein coated plates. 

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Quality Control Assay

The following Quality Control Tests are performed on each new lot to check if they meet the specifications designated for the product:

Luciferase Assay:Considering that the RFs are transcription factors, we adopted luciferase assay method to verify their biological activity in cells. Transfecting luciferase vector that encodes binding sites for the RFs, we measure numerical bioluminescence values after iCP-RFs treatment on the transfected cells. 

Individual lot data can be found on the Product Summary Sheet below:

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