TLR5::GFP

The hTLR5::GFP fusion gene was generated by fusing the green fluorescent protein (GFP) gene to the C terminus of the human Toll-like receptor 5 (TLR5) gene. This fusion protein can be used to study the localization of TLR5. Transfected cells can be analyzed for GFP expression by flow cytometry and by Western-blotting using GFP antibodies. The TLR5-GFP fusion gene is cloned in the pUNO plasmid under the control of the strong and ubiquitous mammalian promoter EF1α/HTLV.

hTLR5::GFP fusion gene has been fully sequenced, its fluorescence confirmed and its function tested in HEK293 cells coexpressing an NF-κB reporter plasmid (pNiFty-SEAP).

Specifications

Human TLR5::GFP fusion gene (3429 bp)

Selectable with Blasticidin in both E. coli and mammalian cells

pUNO1 Contents

• 20 µg of lyophilized DNA
• 2 x 1 ml blasticidin at 10 mg/ml

Product is shipped at room temperature.

Lyophilized DNA should be stored at -20°C. Upon receipt, store blasticidin at 4°C or -20°C.

Resuspended DNA should be stored at -20°C and is stable for up to 1 year.

Blasticidin is a harmful compound. Refer to the safety data sheet for handling instructions.

Details

Toll-like receptor 5 (TLR5) is the Toll-like molecule that recognizes flagellin from both Gram-positive and Gram-negative bacteria. TLR5 was identified by the presence of the TIR domain and is expressed in spleen, peripheral blood leukocytes and epithelial cells. Activation of the receptor stimulates the production of pro-inflammatory cytokines, such as TNF-α, through signaling via the adaptor protein MyD88 and the serine kinase IRAK [1, 2]. TLR5 can generate a pro-inflammatory signal as a homodimer suggesting that it might be the only TLR required for flagellin recognition [2].

The hTLR5::GFP fusion gene was generated by fusing at the C terminus of the human TLR5 gene to a GFP variant. A synthetic intron was added between both moieties to increase the activity of GFP. This hybrid protein absorbs blue light (major peak at 480 nm) and emits green light (major peak at 505 nm).

The hTLR5::GFP fusion gene is under the control of the strong and ubiquitous hEF1/HTLV promoter. This composite promoter comprises the Elongation Factor-1α (EF-1α) core promoter [3] and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat [4]. The SV40 late polyadenylation signal enables efficient cleavage and polyadenylation reactions resulting in high levels of steady-state mRNA.

1. Gewirtz AT. et al. (2001). J Immunol, 167(4):1882-5.
2. Hayashi F. et al. (2001). Nature, 410(6832):1099-103.
3. Kim et al. (1990). Gene 2: 217-223.
4. Takebe et al. (1988). Mol. Cell Biol. 1: 466-472.