Mammalian development requires the specification of over 200 unique cell types from a single totipotent cell. Embryonic stem (ES) cells are derived from the inner cell mass of the developing blastocyst and can be propagated in culture in an undifferentiated state while maintaining the capacity to generate any cell type in the body. The recent derivation of human ES cells provides a unique opportunity to study early development and an understanding of the transcriptional regulatory circuitry that is responsible for pluripotency and self-renewal in human ES cells is fundamental to understanding human development and realizing the therapeutic potential of these cells.
Three transcription factors are known to be critical in the maintenance of ESC pluripotency: Oct4, Nanog, and Sox2. Oct4 (Pou5f1) has a highly conserved role in maintaining pluripotent cell populations (Nichols et al., 1998; Morrison and Brickman, 2006), and its expression level dictates ESC fate (Niwa et al., 2000). SOX2 forms a complex with OCT4 and is necessary to cooperatively activate target genes in ESCs (Yuan et al., 1995; Ambrosetti et al., 1997). These factors comprise one essential circuit regulating ESC pluripotency in which OCT4 regulates Sox2, and additionally, the OCT4-SOX2 complex activates Oct4 expression (Okumura-Nakanishi et al., 2005).
The transcription factors OCT4, SOX2, and NANOG have essential roles in early development and are required for the propagation of undifferentiated embryonic stem (ES) cells in culture.
SBI has built lentivector-based transciptional Response Reporters that are responsive to Oct4 or Sox2 activity as response elements or Promoter Reporters for either Human or Mouse Oct4 and Nanog into the pGreenZeo, pRedZeo and pRedTK systems.
Choice of fluorescent screening marker The lenti-reporter systems employ either a Green Fluorescent Protein (copGFP) or a Red Fluorescent Protein (RFP). Depending upon your screen, either Green or Red markers may be suitable and compatible with other fluorescent protein markers in your experimental scheme. The Response Reporters feature the dual reporting system with both GFP and Luciferase.
Choice of antibiotic selection marker The Response Reporters have an optional constitutive EF1-Puro or -Neo selection cassette for establishing cells lines. The Promoter Reporters feature Zeomycin (Zeo) resistance marker confers resistance to the antibiotic Zeocin™. Zeocin™ causes cell death by intercalating into DNA and causes DNA strand breakage. This antibiotic is effective on most aerobic cells and is therefore useful for selection in bacteria, eukaryotic microorganisms, plant and animal cells. Thymidine kinase (TK) is a Herpes simplex virus-derived enzyme that acts on the guanosine nucleotide analog ganciclovir. TK phosphorylates ganciclovir, and TK+ cells produce highly-toxic triphosphates that lead to cell death (negative selection).
All Stem Cell Pluipotency Monitors are available as lentivector plasmids or as pre-packaged lentiviral preparations (using the HIV lentiviral backbone and pseudotyped with VSV-G protein)—ready for transduction of target cells. The standard product size is 10 ug of plasmid or 1 x 10^6 IFUs per packaged lentiviral reporter. Positive and negative transduction control plasmids and pre-packaged viral particles are also available.
Monitor and Quantitate the Pluripotent State
Track Pluripotency in Living Cells with GFP and Quantitate Activities of Oct4 or Sox2 with Luciferase - all built into the Dual pGreenFire Response Reporters
Oct4 (Cr4) Response reporters: Catalog #s SR20070 series Sox2 (SRR2) Response reporters: Catalog #s SR20071 series
Verify the pluripotent state of your stem cells using Oct4 conserved enhancer (CR4) or Sox2 (SRR2) response reporters. These tools allow for the easy and accurate monitoring of ES and iPS cell pluripotency. The CR4 and SRR2 response reporters work for both Human and Mouse stem cells and enable more specific monitoring of Oct4 and Sox2 activities. These response reporters feature both GFP and Luciferase for quantitative transactivation assays.
All Pluipotency Response Reporters are available as lentivector plasmids or as pre-packaged lentiviral preparations (using the HIV lentiviral backbone and pseudotyped with VSV-G protein)—ready for transduction of target cells. The standard product size is 10 ug of plasmid or 1 x 10^6 IFUs per packaged lentiviral reporter. Positive and negative transduction control plasmids and pre-packaged viral particles are also available.
Sample data in Embryonic Stem Cells
All Stem Cell Pluipotency Monitors are available as lentivector plasmids or as pre-packaged lentiviral preparations (using the HIV lentiviral backbone and pseudotyped with VSV-G protein)—ready for transduction of target cells. The standard product size is 10 ug of plasmid or 1 x 10^6 IFUs per packaged lentiviral reporter. Positive and negative transduction control plasmids and pre-packaged viral particles are also available.
Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem. 2005 Feb 18;280(7):5307-17.
Tomioka M, Nishimoto M, Miyagi S, Katayanagi T, Fukui N, Niwa H, Muramatsu M, Okuda A. Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res. 2002 Jul 15;30(14):3202-13.
Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Schöler H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998 Oct 30;95(3):379-91.
Morrison GM, Brickman JM. Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development. Development. 2006 May;133(10):2011-22.
Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000 Apr;24(4):372-6.
Yuan H, Corbi N, Basilico C, Dailey L. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev. 1995 Nov 1;9(21):2635-45.
Ambrosetti DC, Basilico C, Dailey L. Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites. Mol Cell Biol. 1997 Nov;17(11):6321-9.
Okumura-Nakanishi S, Saito M, Niwa H, Ishikawa F. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J Biol Chem. 2005 Feb 18;280(7):5307-17.
