General descriptionThe Human STEMCCA Cre-Excisable Constitutive Polycistronic (OKSM) Lentivirus Kit contains high titer polycistronic (OKSM) lentivirus and Polybrene transfection reagent that have been validated for the generation of human induced pluripotent stem cells from human foreskin fibroblasts (HFFs). The Cre-excisable STEMCCA vector, comprised of the transcription factors human Oct-4, Klf4, SOX-2, and c-Myc (OKSM) separated by the self-cleaving 2A peptide and IRES sequences, includes flanking LoxP sites that enable Cre-mediated excision of the viral transgenes following reprogramming (Sommer CA, 2009).
The use of a single polycistronic lentiviral vector significantly improves reprogramming efficiencies and reduces the number of viral integrations. Furthermore, the ability to remove the reprogramming vector has been shown to improve the developmental potential of iPS cells and significantly increase their capacity to undergo directed differentiation in vitro (Sommer CA, 2010).
Preparation NoteLentivirus is stable for at least 6 months when stored at -80°C. After first thaw, place immediately on ice and store in working aliquots to avoid further freeze thaws. Avoid freeze thaws as this will result in a decrease in the virus titer. Polybrene transfection reagent is stable for at least 1 year when stored at -20°C.
Analysis NoteTested 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 Notes1. Human STEMCCA Cre-Excisable Constitutive (OKSM) Lentivirus: (Part number SCR545-1) Two (2) vials of EF1α-hSTEMCCA-LoxP (OKSM) Lentivirus (Part number CS204503). Each vial contains 15 µL of high titer lentivirus. 2. Polybrene Transfection Reagent: (Part number TR-1003-50UL) One (1) vial containing 50 µL of 10 mg/mL stock.
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