Human TLR7 NF-kB/SEAP - (SEAPorter™) Stable Reporter Cell Line

Product Discontinued

Human TLR7 NF-kB/SEAP - (SEAPorter™) Stable Reporter Cell Line Summary

• Description: The TLR7 reporter line can be used for TLR7-dependent functional assays as well as screening of TLR7 agonists or antagonists. The TLR7 reporter cell line has been validated by flow cytometry (fig. 2) and ligand dose response assay (fig. 1).
  
  Contents: 3~4 x 10^6 cells
  Biosafety Level: 2
• Immunogen: The TLR7 reporter cell line is a stably co-transfected cell line which expresses full-length human Toll-like receptor7 (TLR7) and the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NF-kB response element.
• Target Species: Human
• Specificity: TLR7/NF-kB/SEAP
• Selection Agent: Blasticidin and G418.
• Growth Properties: Adherent Morphology: Epithelial
• RCL Type: NF-kB/SEAP - (SEAPorter™)
• Reporter Gene: Secreted alkaline phosphatase (SEAP)
• Host: HEK293
• Gene: TLR7

Applications/Dilutions

• Dilutions:
  • Flow Cytometry
  • In vitro assay
  • Ligand Activation
• Application Notes: Use in In vitro assay reported in scientific literature (PMID 26193334)
• Agonist:
  • Imidazoquinoline Resiquimod (R-848), TLR7 and TLR8 ligand NBP2-26231
• Readout System:
  • Secreted Alkaline Phosphatase Reporter Assay Kit (Colorimetric) NBP2-25285
  • Intracellular Staining Kit NBP2-26248

Packaging, Storage & Formulations

• Storage: Store in gas phase of liquid nitrogen.
• Reconstitution Instructions: Growth Medium: DMEM with 4.5 g/L glucose + 10% FBS + 4 mM L-glutamine + 1 mM sodium pyruvate + 100 units/ml penicillin + 0.1 mg/ml streptomycin + 10 ug/ml blasticidin + 0.5 mg/ml G418 (Geneticin).

Details for Array

• Type: Cell

Notes

Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should be observed.
- Use pipette aids to prevent ingestion and keep aerosols down to a minimum.
- No eating, drinking or smoking while handling the TLR7 reporter line.
- Wash hands after handling the TLR7 reporter line and before leaving the lab.
- Decontaminate work surface with disinfectant or 70% ethanol before and after working with cells.
- All waste should be considered hazardous.
- Dispose of all liquid waste after each experiment and treat with bleach.

Alternate Names for Human TLR7 NF-kB/SEAP - (SEAPorter™) Stable Reporter Cell Line

• TLR7
• toll-like receptor 7

Background

Toll-like receptor 7 (TLR7) is a type I transmembrane protein expressed on the surface of endosomes and has a role in pathogen-associated molecular patterns (PAMPs) recognition and host defense (1-3). TLR7 is primarily expressed in the brain, placenta, spleen, stomach, and lungs (4). TLR7 recognizes microbial single stranded RNA (ssRNA), specifically guanosine and its derivatives (1-3). Human TLR7 cDNA encodes a 1049 amino acid (aa) protein with a theoretical molecular weight (MW) of 120.9 kDa (4). The TLR7 protein consists of a signal sequence, an 813 aa extracellular domain containing leucine-rich repeats (LRRs) which form a horseshoe-like shape, a 21 aa transmembrane domain, and a 189 aa cytoplasmic domain with cytosolic Toll-interleukin-1 receptor homology (TIR) domains (1,2,4). TLR7 and its fellow subfamily members, TLR8 and TLR9, possess a characteristic Z-loop between two LRRs with proteolytic Z-loop processing required for TLR activation (2). Z-loop cleavage in TLR7 allows for guanosine and uridine-rich ssRNA binding to the 1st and 2nd ligand binding site, respectively (2). The TIR domain associates with the adaptor protein myeloid differentiation primary response protein (MyD88) to initiate downstream signaling (1-3,5,6). Following activation by PAMPs, TLR7 dimerizes and bound MyD88 interacts with interleukin-1 receptor-associated kinase-4 (IRAK-4) (1,5). Together the complex recruits IRAK-1 and IRAK-2, which become phosphorylated, and interact with tumor necrosis factor receptor-associated factor 6 (TRAF6) (1,5). TRAF6 induces the activation of mitogen-activated protein kinase (MAPK), nuclear factor-kappaB (NF-kappaB), and interferon-regulatory factor 7 (IRF7), leading to interferon production and pro-inflammatory cytokine secretion associated with immune response (1,5).

While TLRs play an important role in innate immune response, dysfunction in the TLR-MyD88 signaling cascade has also been reported in various autoimmune disorders (5,6). Elevated expression of TLR7 is associated with increased risk of system lupus erythematosus (SLE), an autoimmune disease involving B cell hyperactivity (6,7). Studies involving mouse models has also found that increased TLR7 expression predisposes mice to a lupus-like disease (7). Therapeutics targeting TLR7 have been developed to either enhance or inhibit its activity depending on the circumstance. For example, TLR7 agonists such as imiquimod, resiquimod, and 852A are used to increase TLR7 activity for treatment of cancers and to fight viral infections (7,8). On the other hand, TLR7 antagonists inhibit its activation and have been developed to combat chronic immune stimulation as seen in inflammatory and autoimmune diseases (8).

References

1. Petes C, Odoardi N, Gee K. The Toll for Trafficking: Toll-Like Receptor 7 Delivery to the Endosome. Front Immunol. 2017;8:1075. https://doi.org/10.3389/fimmu.2017.01075

2. Maeda K, Akira S. TLR7 Structure: Cut in Z-Loop. Immunity. 2016;45(4):705-707. https://doi.org/10.1016/j.immuni.2016.10.003

3. Krieg AM, Vollmer J. Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunol Rev. 2007;220:251-269. https://doi.org/10.1111/j.1600-065X.2007.00572.x

4. Uniprot (Q9NYK1)

5. Zheng C, Chen J, Chu F, Zhu J, Jin T. Inflammatory Role of TLR-MyD88 Signaling in Multiple Sclerosis. Front Mol Neurosci. 2020;12:314. https://doi.org/10.3389/fnmol.2019.00314

6. Chi H, Li C, Zhao FS, et al. Anti-tumor Activity of Toll-Like Receptor 7 Agonists. Front Pharmacol. 2017;8:304. https://doi.org/10.3389/fphar.2017.00304

7. Fillatreau S, Manfroi B, Dorner T. Toll-like receptor signalling in B cells during systemic lupus erythematosus. Nat Rev Rheumatol. 2021;17(2):98-108. https://doi.org/10.1038/s41584-020-00544-4

8. Patinote C, Karroum NB, Moarbess G, et al. Agonist and antagonist ligands of toll-like receptors 7 and 8: Ingenious tools for therapeutic purposes. Eur J Med Chem. 2020;193:112238. https://doi.org/10.1016/j.ejmech.2020.112238

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Reporter Cell Lines are guaranteed for 1 year from date of receipt.

Publications for TLR7 Reporter Cell Line (NBP2-26278)(6)

Publications using NBP2-26278

• Miller Sm, Cybulski V, Whitacre M et Al. Novel Lipidated Imidazoquinoline TLR7/8 Adjuvants Elicit Influenza-Specific Th1 Immune Responses and Protect Against Heterologous H3N2 Influenza Challenge in Mice Front Immunol 2020-03-10 [PMID: 32210973]
• Shinchi H, Crain B, Yao S et al. Enhancement of the immunostimulatory activity of a TLR7 ligand by conjugation to polysaccharides Bioconjug. Chem. 2015-07-20 [PMID: 26193334] (In vitro)
• Hayashi T, Yao S, Crain B et al. Induction of Tolerogenic Dendritic Cells by a PEGylated TLR7 Ligand for Treatment of Type 1 Diabetes PLoS ONE. 2015-06-16 [PMID: 26076454]
• Zurcher C, Sauter KS, Schweizer M. Pestiviral Erns blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA. Veterinary Microbiology. 2014-10-13 [PMID: 25457366]
• Chan M, Hayashi T, Mathewson RD et al. Identification of substituted pyrimido[5,4-b]indoles as selective Toll-like receptor 4 ligands. J Med Chem. 2013-06-13 [PMID: 23656327]
• Greene CM, Carroll TP, Smith SG et al. TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells. J Immunol. 2005-02-01 [PMID: 15661927]
  
  Details:
  Functional Assays: 1.TLR2/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-102). 2. TLR4/MD-2/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-104MD2). 3. TLR4/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-104).4.TLR7/NF-kB/SEAP Stably Transfected HEK 293 Cells (IML-107). 5. LPS from E. Coli, TLR4 Ligand (IMG-2204). 6. SEAPorter Assay Kit (10055K).

FAQs for TLR7 Reporter Cell Line (NBP2-26278). (Showing 1 - 1 of 1 FAQs).

1. What is the construct for this cell line and how many NF-Kb binding sites are there?
   • The only data I am able to find on this particular cell line is in the publications listed on the datasheet: TLR7 Reporter Cell Line (NBP2-26278) Publications Section. Unfortunately, we cannot offer the construct for this cell line. This cell line is proprietary to Novus Biologicals, and I am not able to offer any further information. I apologize for any inconvenience.

Bioinformatics

• Gene Symbol: TLR7