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Comparison of bovine leukemia virus (BLV) and CMV promoter-driven reporter gene expression in BLV-infected and non-infected cells - PubMed

  • ️Thu Jan 01 2004

Comparison of bovine leukemia virus (BLV) and CMV promoter-driven reporter gene expression in BLV-infected and non-infected cells

Jerome S Harms et al. Genet Vaccines Ther. 2004.

Abstract

BACKGROUND: Viral promoters are used in mammalian expression vectors because they generally have strong activity in a wide variety of cells of differing tissues and species. METHODS: The utility of the BLV LTR/promoter (BLVp) for use in mammalian expression vectors was investigated through direct comparison to the CMV promoter (CMVp). Promoter activity was measured using luciferase assays of cell lines from different tissues and species stably transduced with BLVp or CMVp driven luciferase vectors including D17, FLK, BL3.1 and primary bovine B cells. Cells were also modified through the addition of BLV Tax expression vectors and/or BLV infection as well as treatment with trichostatin A (TSA). RESULTS: Results indicate the BLV promoter, while having low basal activity compared to the CMV promoter, can be induced to high-levels of activity similar to the CMV promoter in all cells tested. Tax or BLV infection specifically enhanced BLVp activity with no effect on CMVp activity. In contrast, the non-specific activator, TSA, enhanced both BLVp and CMVp activity. CONCLUSION: Based on these data, we conclude the BLV promoter could be very useful for transgene expression in mammalian expression vectors.

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Figures

Figure 1
Figure 1

Schematic representation of BLV promoter used in comparison studies. The BLV promoter (BLVp) consisting of the U3 region of the 5'LTR of BLV includes the basic elements of transcription start site (+1), CAAT (nt -97/-92) and TATA (nt -43/-37) boxes as shown. Unique to the BLVp are the three imperfectly conserved 21 bp sequences known as the Tax Responsive Elements (TxRE). The numbers following the TxRE designation represent its position relative to the transcription start site. Each TxRE contains a consensus E box-binding motif overlapping an imperfect cyclic AMP responsive element motif (CRE/Ebox). Additionally, the BLVp contains a glucocorticoid responsive element (GRE), Nuclear Factor Kappa Binding motif (NFkB), and B cell specific PU.1 or Spi-B transactivator binding motif (PU.1/Spi-B). The transcription elements are not drawn to scale.

Figure 2
Figure 2

BLVp and CMVp activity comparison in D17, FLK, primary cow B cells, and BL3.1. Relative light units (RLU) of luciferase activity driven by either the BLV promoter (BLVp) or CMV promoter (CMVp) of 1 × 106 stably transduced cells was measured during a 10 s period. Bars represent the arithmetic mean and variance of 10 experiments. *P < 0.05; **P < 0.001 determined by t-test.

Figure 3
Figure 3

BLV infection enhances BLVp activity but has no effect on CMVp activity. D17 cells or D17 cells infected with and productively expressing BLV (D17+BLV) were transduced with luciferase expression vectors. Relative light units (RLU) of luciferase activity driven by either the BLV promoter (BLVp) or CMV promoter (CMVp) of 1 × 106 stably trasduced cells was measured during a 10 s period. Bars represent the arithmetic mean and variance of 10 experiments. **P < 0.001 determined by t-test.

Figure 4
Figure 4

BLV Tax expression significantly enhances BLVp activity but has no effect on CMVp activity. D17 cells and primary bovine B cells (D17; B cells), or D17 cells and primary bovine B cells stably transduced with a BLV Tax expression vector (D17+TAX; B cells+TAX), were assayed. Relative light units (RLU) of luciferase activity driven by either the BLV promoter (BLVp) or CMV promoter (CMVp) of 1 × 106 stably transduced cells were measured during a 10 s period. Bars represent the arithmetic mean and variance of 10 experiments. **P < 0.001 determined by t-test.

Figure 5
Figure 5

Tax trans-gene expression significantly enhances BLVp activity in cells producing high levels of BLV. FLK and BL3.1 cells, or FLK and BL3.1 cells stably transduced with a BLV Tax expression vector (FLK+TAX; BL3.1+TAX) were assayed. Relative light units (RLU) of luciferase activity driven by either the BLV promoter (BLVp) or CMV promoter (CMVp) of 1 × 106 stably transduced cells were measured during a 10 s period. Bars represent the arithmetic mean and variance of 10 experiments. *P < 0.05; **P < 0.001 determined by t-test.

Figure 6
Figure 6

Trichostatin A (TSA) enhances BLVp and CMVp activity. Relative light units (RLU) of luciferase activity driven by either the BLV promoter (BLVp) or CMV promoter (CMVp) of 1 × 106 stably trasduced cells was measured during a 10 s period. BL3.1 cells were either non-treated or treated with 500 nM TSA for 48 h. Bars represent the arithmetic mean and variance of 10 experiments. **P < 0.001 determined by t-test.

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