Human TLR9 Reporter HEK293 Cells

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SEAP reporter system in HEK-Blue™ TLR9 cells

SEAP reporter 293 cells expressing the human TLR9 gene

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-Blue™ hTLR9 cells

HEK-Blue™ hTLR9 cells overexpress the human TLR9 (hTLR9) gene. They also express a SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of an NF-κB inducible promoter comprised of an IFN-β minimal promoter fused to five NF-κB and AP-1 binding sites. Levels of SEAP in the supernatant can be easily determined with HEK-Blue™ Detection, a cell culture medium that allows real-time detection of SEAP activity.

Unlike their parental cells, HEK-Blue™ Null1,  which weakly express TLR9, HEK-Blue™ hTLR9 cells are responsive to stimulation with TLR9 agonists, such as oligonucleotides containing CpG motifs (CpG ODNs). As expected, hTLR9 overexpression in HEK-Blue™ hTLR9 cells allows potent NF-κB responses upon incubation with human-preferred, but not mouse-preferred, CpG-ODNs of class A (e.g. ODN 2216) and class B (e.g. ODN 2006). Of note, HEK-Blue™ hTLR9 cells are more responsive to the class B, human-preferred, ODN 2006. These cells also respond to ODN 2395, a human/mouse-preferred class C (see Figures). 

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 [1-3]. TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs) [1,3].

 More details

Key features:

• Verified overexpression of TLR9 gene by RT-PCR
• Functionally validated with a selection of TLR9 agonists
• Readily assessable SEAP 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. 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.

Figures

HEK-Blue™ hTLR9 cells were stimulated with various TLR and NOD agonists: Pam3CSK4 (100 ng/ml), Poly(I:C) (50 ng/ml), LPS-EB ultrapure (100 ng/ml), recombinant flagellin from S. typhimurium (10 ng/ml), CL264 (1 µg/ml), CL097 (1 µg/ml), ssRNA40/LyoVec™ (5 µg/ml), ODN 2006 (10 µg/ml), C12-iE-DAP (100 ng/ml ), L18-MDP (100 ng/ml ), and TNF-α (100 ng/ml ). After 18h incubation (24h incubation for CL264, C12-iE-DAP and L18-MDP ligands), NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ and reading the OD at 655 nm.

HEK-Blue™ hTLR9 and HEK-Blue™ Null1 (control) cells were incubated in HEK-Blue™ Detection medium and stimulated with 30 µg/ml ODN2216 (human specific, type A), 0.3 µg/ml ODN2006 (human specific, type B), 30 µg/ml ODN2395 (human specific, type C), 30 µg/ml ODN1826 (mouse specific, type B), or 5 µg/ml E.coli ssDNA/LyoVec™. After 18h incubation, NF-kB-induced SEAP activity was assessed using QUANTI-Blue™ and reading the OD at 655 nm.

HEK-Blue™ hTLR9 cells were stimulated with increasing concentrations of TLR9 agonists. In the top graph, the response to ODN2006 (human specific, type B) is shown. In the bottom graph, the response to ODN2216 (human specific, type A) and ODN2395 (human/mouse, type C) is presented. After 18h incubation, NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. The response ratio was calculated by dividing the OD at 655 nm for the treated cells by the OD at 655 nm for the untreated cells.

 

Specifications

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

Antibiotic resistance: Blasticidin, Zeocin®

Quality Control:

• Human TLR9 expression has been verified by RT-PCR 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-Blue™ hTLR9 cells in a cryovial or shipping flask
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml)
• 1 pouch of HEK-Blue™ Detection (cell culture medium for real-time detection of SEAP)

 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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