pNFkB-DD-tdTomato載體描述 pNFkB-DD-tdTomato is a reporter vector that allows you to monitor NFkB activation in mammalian cells. The vector contains an NFkB enhancer element (composed of four tandem copies of the NFkB consensus binding sequence; 1) upstream of a minimal TA promoter (PTA), which consists of the TATA box from the herpes simplex virus thymidine kinase (HSV-TK) promoter. The vector encodes the reporter protein DD-tdTomato, a ligand-dependent, destabilized red fluorescent protein that minimizes background fluorescence from leaky promoters.
tdTomato is a member of the family of fruit fluorescent proteins derived from the Discosoma sp. red fluorescent protein, DsRed (excitation and emission maxima: 554 and 581 nm, respectively; 1, 2). DD-tdTomato is a modified version of tdTomato that is tagged on its N-terminus with the ProteoTuner? destabilization domain (DD; 3). The presence of this destabilization domain causes rapid, proteasomal degradation of the fluorescent fusion protein; however, when the membrane permeant ligand Shield1 is added to the medium, it binds to the destabilization domain and protects the fusion protein from degradation.
In the absence of Shield1, the destabilization domain causes the degradation of any DD-tdTomato reporter protein produced prior to promoter activation, thus reducing background fluorescence. In order to analyze NFκB activation, an inducer of choice is added to the medium along with the Shield1 stabilizing ligand, which effectively stabilizes the reporter protein, allowing it to accumulate. As a result, only the reporter molecules expressed during promoter induction will contribute to the fluorescence signal, providing a considerably higher signal-to-noise ratio than that obtained with non-destabilized or constitutively destabilized reporter systems. The high signal-to-noise ratio also allows the monitoring of NFκB activation during discrete windows of time when Shield1 is added to the cell medium for discrete periods of time.
The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418 (4). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
The pNFkB-DD-tdTomato Reporter vector, available as part of the NFkB DD Red Reporter System (Cat. No. 631081), can be used to monitor NFkB activation in live cells as well as in vivo. pNFkB-DD-tdTomato can be transfected into mammalian cells using any standard transfection method. If required, stable transfectants can be selected using G418.
Propagation in E. coli
Recommended host strains: DH5α?, HB101, and other general purpose strains. Single-stranded DNA production
requires a host containing an F plasmid such as JM109 or XL1-Blue.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: high
Plasmid incompatibility group: pMB1/ColE1
Excitation and emission maxima of tdTomato
Excitation maximum = 554 nm
Emission maximum = 581 nm References 1. Pessara, U. & Koch, N. (1990) Mol. Cell Biol. 10(8):4146–4154.
2. Shaner, N. C., et al. (2004) Nature Biotech. 22(12):1567-1572.
3. Campbell, R. E. et al. (2002) Proc. Natl. Acad. Sci. USA 99(12):7877–7882.
4. Banaszynski, L. et al. (2006) Cell 126(5):995–1004.
5. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
Note: The vector sequence was compiled from information in the sequence databases, published literature, and other sources, together with partial sequences obtained by Clontech. This vector has not been completely sequenced.
