Human STEMCCA/TAT-Cre Bundle

The 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).

Cre Recombinase is an enzyme from bacteriophage P1 that catalyzes the site-specific recombination between two DNA recognition sites termed loxP sites. The LoxP recognition site consists of two 13 bp inverted repeats flanking a 8 bp spacer region. Because the Cre gene and loxP sites are not native to the genomes of most species, LoxP sites can be engineered and introduced into target cells and thus used as a means to precisely control the expression of genes in vitro (i.e. cultured cells) and in vivo (i.e. animal models).

EMD Millipore′s TAT-CRE Recombinase is a recombinant cell-permeant fusion protein consisting of a basic protein translocation peptide derived from HIV-TAT (TAT), a nuclear localization sequence (NLS), the Cre protein and an N-terminal histidine tag (H6) for efficient purification of the protein from E. coli.

EMD Millipore′s TAT-CRE Recombinase (SCR508) has been shown to effectively excise STEMCCA viral transgenes from both Human and Mouse IPS cells (SCR545).

Convenient kit that contains all reagents necessary to generate transgene-free human IPS cells.

STEMCCA lentivirus 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.TAT-CRE rembombinase is stable for 3 months from date of receipt when stored at -20°C or -80°C. Upon first thaw, centrifuge the vial and gently mix the solution. Aliquot into smaller working volumes and freeze at -20°C or -80°C. Before use, dilute TAT-CRE to the appropriate concentration with culture medium and filter through a 0.2um low protein binding syringe filter (Millipore^® Cat. No. SLGV 033RS or SLGV013 SL).

Each lot of STEMCCA lentivirus have been 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.

Each lot of TAT-CRE Recombinase protein is rigorously quality control tested for the following parameters:
• Purity: single band around 41 kDa with greater than 70% protein purity on an SDS-PAGE gel
• Functional activity: mediates recombination of LoxP-modified alleles in a HEK293T- Cre reporter cell line
• Endotoxin levels: less than 1 EU/ug protein
• Mycoplasma negative

1. 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 uL of high titer lentivirus. 2. Polybrene Transfection Reagent: (Part number TR-1003-50UL) One (1) vial containing 50 uL of 10 mg/mL stock. 3. TAT-CRE Recombinase (Part number SCR508) One (1) vial containing 150 uL of 10,000 Units/mL TAT-Cre Recombinase protein

Millipore is a registered trademark of Merck KGaA, Darmstadt, Germany

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