Human TLR9 Dual-Reporter HEK293 Cells

NF-κB-SEAP/KI-[IL-8]Lucia reporter systems
in HEK-Dual™ hTLR9 cells

Human TLR9 (NF-κB-SEAP / KI-[IL-8]Lucia) dual-reporter HEK293 cells

InvivoGen offers a human embryonic kidney 293 (HEK293)-derived cell line, specifically designed for the study of human TLR9 (Toll-Like Receptor 9)-induced NF-κB signaling pathway:

• HEK-Dual™ hTLR9 cells

HEK-Dual™ hTLR9 cells overexpress the human TLR9 (hTLR9) gene. Like their parental cells, HEK-Dual™ Null, they are knockout of TLR3, TLR5, and TNFR genes (all of which are endogenously expressed in HEK293 cells). They also feature two inducible reporter systems:
— a SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of an NF-κB inducible promoter
— a secreted Lucia luciferase reporter gene placed under the control of the endogenous IL-8 promoter; the coding sequence of IL-8 was replaced by the Lucia luciferase ORF using knock-in technology. IL-8 is a chemokine produced in response to TLR agonists in an NF-κB and AP-1-dependent manner [1, 2].
The two reporter proteins, SEAP and Lucia luciferase can be readily measured in the supernatant by using QUANTI-Blue™ Solution and QUANTI‑Luc™ 4 Lucia/Gaussia, respectively.

Unlike their parental cells which weakly express TLR9, HEK-Dual™ hTLR9 cells are responsive to stimulation with TLR9 agonists, such as oligonucleotides containing CpG motifs (CpG ODNs).

Background:

Toll-Like Receptor 9 (TLR9) is one of the most studied pattern recognition receptors (PRRs) for nucleic acids. It is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [3-5]. TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs) [3,5].

More details

Key features:

• Verified overexpression of TLR9 gene by RT-PCR
• Functionally validated with a selection of TLR9 agonists
• Readily assessable SEAP and Lucia reporter activity

Applications:

• Study of the NF-κB-dependent TLR9 signaling pathway
• Screening of novel specific activators or inhibitors of the TLR9 signaling pathway

References

1. Ohta K. et al., 2014. TLR-mediated interleukin-8 production by human submandibular gland epithelial cells. Mol Med Rep. 10(5):2377-82.
2. Roebuck KA. et al., 1999. Regulation of interleukin-8 gene expression. J Interferon Cytokine Res. 19(5):429-38.
3. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
4. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
5. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.

Figures

Detection of the NF-kB response using QUANTI-Blue™. HEK-Dual™ hTLR9 (NF/IL8) and HEK-Blue™ hTLR9 cells were stimulated with various TLR agonists: ODN2006 (class B CpG ODN, the ligand of choice for hTLR9; 1 μg/ml), ODN1826 (class B CpG ODN, the ligand of choice for murine TLR9; 1 μg/ml), ODN2216 (class A CpG ODN that preferentially binds to hTLR9; 1 μg/ml), Poly(I:C) (TLR3 agonist; 3 μg/ml), FLA-ST (flagellin from S. typhimurium, TLR5 agonist; 100 ng/ml), and TNF-α (10 ng/ml). The TLR3, TLR5 and TNFR activities due to the endogenous expression of these receptors in HEK-Blue™ hTLR9 cells are shown in gray. After 24 hour incubation, NF‑kB‑induced SEAP activity was assessed using QUANTI‑Blue™ and reading the optical density (OD) at 655 nm.

Detection of the IL-8 response using QUANTI-Luc™. HEK-Dual™ hTLR9 (NF/IL8) cells were stimulated with TLR9 agonists: ODN2006 (1 μg/ml), ODN1826 (1 μg/ml) and ODN2216 (1 μg/ml). After 24 hour incubation, activation of the IL-8 promoter was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI‑Luc™, a Lucia luciferase detection reagent. The activation of the IL-8 promoter is expressed as fold increase relative to untreated cells which was calculated by dividing the RLUs for the treated cells by the RLUs for the untreated cells.

Specifications

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Antibiotic resistance: Blasticidin, Zeocin®, and hygromycin

Quality Control:

• Human TLR9 expression has been verified by RT-PCR and functional assays.
• The biallelic replacement of the human interleukin-8 (IL-8) open reading frame (ORF) with the Lucia luciferase reporter ORF was verified by PCR and sequencing.
• The inability to produce IL-8 has been confirmed by ELISA.
• TLR3, TLR5, and TNFR triple knockout has been verified by DNA sequencing and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

InvivoGen's products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ HEK-Dual™ hTLR9 cells in a cryovial or shipping flask
• 1 ml of Hygromycin B Gold (100 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Details

Toll-Like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [3,4]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [1,3].

TLR9 is mainly expressed in subsets of Dendritic Cells and in B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [3]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [3]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [5].
 

 Get more information about CpG-ODNs Classes.

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
4. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
5. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.

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