Mouse TLR9 Reporter HEK293 Cells

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SEAP reporter system in HEK-Blue™ TLR9 cells

SEAP reporter 293 cells expressing the mouse TLR9 gene

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

• HEK-Blue™ mTLR9 cells

HEK-Blue™ mTLR9 cells overexpress the mouse TLR9 (mTLR9) 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™ mTLR9 cells are responsive to stimulation with TLR9 agonists, such as oligonucleotides containing CpG motifs (CpG ODNs). As expected, mTLR9 overexpression in HEK-Blue™ mTLR9 cells allows potent NF-κB responses upon incubation with mouse-preferred CpG-ODNs of class A (e.g. ODN 1585) and class B (e.g. ODN 1826, ODN 1668). Of note, HEK-Blue™ mTLR9 cells are more responsive to mouse-preferred CpG-ODNs of class B than class A (see Figures). These cells also respond to ODN 2395, a human/mouse-preferred class C. 

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™ mTLR9 and HEK-Blue™ Null1 cells (control cell line) were stimulated with various TLR and NOD agonists: Pam3CSK4 (TLR2 agonist; 100 ng/ml), Poly(I:C) (TLR3 agonist; 1 µg/ml), LPS-EB ultrapure (TLR4 agonist; 100 ng/ml), FLA-ST (flagellin from S. typhimurium, a TLR5 agonist; 100 ng/ml), R848 (TLR7/8 agonist; 1 µg/ml), ODN 1668 (class B ODN specific for mTLR9; 10 µg/ml), ODN 1826 (class B ODN specific for mTLR9; 10 µg/ml), ODN 2006 (class B ODN with a preference for human TLR9; 10 µg/ml), Tri-DAP (NOD1 agonist; 1 µg/ml), MDP (NOD2 agonist; 1 µg/ml), and TNF-α (10 ng/ml). After a 24h incubation, NF‑kB‑induced SEAP activity was assessed using HEK‑Blue™ Detection and reading the optical density (OD) at 655 nm. TNF-α has been included as a positive control. Non‑induced cells (NI) have been included as negative controls.

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:

• Mouse 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™ mTLR9 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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