pRetroX-Tight-Pur-Lu載體描述 pRetroX-Tight-Pur-Luc is a retroviral control vector that expresses firefly luciferase under the control of PTight, a modified Tet-responsive promoter. PTight consists of seven direct repeats of a 36 bp regulatory sequence that contains the 19 bp tet operator sequence (tetO), and a modified minimal CMV promoter (1). This vector is supplied with our Retro-X?Tet-On? and Tet-Off? Advanced Inducible Expression Systems (Cat. Nos. 632104 and 632105). These systems provide ready access to the inducible gene expression strategy of Gossen & Bujard, and incorporate major improvements described by Urlinger, et al. (2-6).
pRetroX-Tight-Pur-Luc is optimized to eliminate promoter interference through LTR self-inactivation. The hybrid 5’ LTR consists of the cytomegalovirus (CMV) type I enhancer and the mouse sarcoma virus (MSV) promoter. This promoter drives high levels of viral genome transcription in HEK 293-based packaging cell lines due, in part, to the presence of adenoviral E1A (7-8) in these cells. The self-inactivating feature of the vector is provided by a deletion in the 3’ LTR enhancer region (U3). During reverse transcription of the retroviral RNA, a copy of the inactivated 3’ LTR U3 region replaces the corresponding region of the 5’ LTR, resulting in the inactivation of the 5’ LTR CMV enhancer sequence. This mechanism can reduce the phenomenon known as promoter interference (9-10) and allow more efficient expression. In this way, pRetroX-Tight-Hyg-Luc supports high viral titers, yet eliminates potential downstream interference between the inducible PTight promoter and its adjacent viral LTR.
pRetroX-Tight-Pur-Luc contains all of the necessary viral RNA processing elements; these include the 5’ and 3’ LTRs, the packaging signal (Ψ+), and the tRNA primer binding site. For safety reasons, however, the vector lacks the structural genes (gag, pol, and env) necessary for retroviral particle formation and replication. pRetroX-Tight-Pur-Luc contains a puromycin resistance gene (Purr) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transfectants. In addition, the vector contains a ColE1 origin of replication and an E. coli Ampr gene for propagation and selection in bacteria.pRetroX-Tight-Pur-Luc can be used as a control vector for either plasmid or retroviral expression vectors. When used as a control for retroviral expression vectors, it must be transfected into a packaging cell line, such as GP2-293; we recommend the Retro-X Universal Packaging System (Cat. No. 631530). Packaging cell lines allow you to produce infectious, replication-incompetent retroviral particles. These retroviral particles can infect a wide range of target cells, but they cannot replicate within these cells due to the absence of viral structural genes. The separate introduction and integration of the structural genes into the packaging cell line minimizes the chance of producing replication-competent virus due to recombination events during cell proliferation.
Propagation in E. coli
Suitable host strains: DH5α?, DH10B?, and other general purpose strains.
Selectable marker: plasmid confers resistance to ampicillin (100 μg/ml) in E. coli hosts.
E. coli replication origin: ColE1
Copy number: high
Notes:
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.
The viral supernatants produced by this retroviral vector could contain potentially hazardous recombinant virus. Due caution must be exercised in the production and
handling of recombinant retrovirus. Appropriate NIH, regional, and institutional guidelines apply. References 1. pTRE-Tight Vectors (April 2003) Clontechniques XVIII(3):13-14.
2. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci USA 89(12):5547–5551.
3. Gossen, M., et al. (1995) Science 268(5218):1766–1769.
4. Urlinger, S. et al. (2000) Proc. Natl. Acad. Sci. USA 97(14):7963-7968.
5. Inducible Gene Expression Systems (January 2007) Clontechniques XXII(1):1-2.
6. Tet-On Advanced Inducible Gene Expression System (2006) Clontechniques XXI(2):1-3.
7. Ory, D.S. et al. (1996) Proc. Natl. Acad. Sci. USA 93(21):11400-11406.
8. Pear, W.S. et al. (1993) Proc. Natl. Acad. Sci. USA 90(18):8392-8396.
9. Barton, G.M. & Medzhitov R. (2002) Proc. Natl. Sci. USA 99(23):14943-14945.
10. Emerman, M. & Temin, H.M. (1984) Cell 39(3 pt. 2):449-467.
