dsDNA-EC

Double-stranded genomic DNA from E. coli K12

dsDNA-EC is an ultrapure preparation of double-stranded DNA from Escherichia coli K12.

Transfected dsDNA-EC is recognized by cytosolic DNA sensors (CDSs) such as cyclic GMP-AMP synthase (cGAS, cGAMP synthase) leading to the production of type I interferons through the STING-TBK1-IRF3 pathway. It is also recognized by the endoplasmic receptor Toll-like receptor 9 (TLR9).

Recognition of dsDNA-EC by CDSs can be studied in THP1-derived reporter cells such as THP1-Blue™ ISG cells, an IRF-SEAP reporter cell line.

Recognition of dsDNA-EC by TLR9 can be studied in HEK-Blue™ TLR9 cells, which are HEK293-derived cells that stably express TLR9 and an NF-kB-SEAP reporter construct.

Specifications

Specificity: CDS agonist and TLR9 agonist.

Working Concentration:

• 30 ng - 1 μg/ml transfected dsDNA-EC to activate CDSs
• 1 μg/ml transfected dsDNA-EC to activate TLR9

Endotoxin level:  < 0.01 EU/µg.

Solubility: 1 mg/ml in water.

Contents

• 200 μg lyophilized dsDNA-EC (double-stranded DNA from E. coli K12)
• 1.5 ml endotoxin-free water

dsDNA-EC is shipped at room temperature.

Stored at -20˚C.

Lyophilized product is stable for 1 year when properly stored.

Description

Intracellular DNA is recognized by the innate immune system via the endosomal TLR9 receptor and multiple cytosolic DNA sensors (CDSs) which display contextual preferences for the recognition of DNA [1].

Unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs) [2], present on bacterial DNA, are recognized by TLR9 [3] which subsequently engages an intracellular pathway leading to NF-κB activation. CDS ligands, including transfected dsDNA-EC, trigger type I interferon (IFN) production and the induction of IFN-stimulated genes (ISG) through interferon regulatory factors (IRFs). In order to facilitate their study, InvivoGen has developed stable reporter cells in two well established immune cell models, the human monocytic THP-1 cell line and the murine RAW 264.7 macrophages. The human monocytic cell line THP-1 has been shown to express all the CDSs [4-6], with the exception of DAI [7]. The RAW 264.7 macrophages have been reported to express several CDSs; IFI16 [8], DDX41 and AIM29.These cells express a reporter gene, either SEAP (secreted embryonic alkaline phosphatase) or Lucia, a secreted luciferase, under the control of an IRF-inducible promoter.

1. Sharma S. & fitzgerald Ka. 2011. Innate immune sensing of DNA. PLoS Pathog. 7(4):e1001310.
2. Krieg, a.m. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature, 374(6522):546-9.
3. Krug a. et al., 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol, 31(7): 2154-63.
4. unterholzner L. et al., 2010. IFI16 is an innate immune sensor for intracellular DNA. Nat Immunol. 11(11):997-1004.
5. Zhang Z. et al., 2011. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol.12(10):959-65.
6. arakawa r. et al., 2010. Characterization of LRRFIP1. Biochem Cell Biol. 88(6):899-906.
7. Lippmann J. et al., 2010. IFNbeta responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI). Cell Microbiol. 10(12):2579-88.
8. Kawasaki t. et al., 2011. Recognition of nucleic acids by pattern-recognition receptors and its relevance in autoimmunity.. Immunol Rev. 243(1):61-73.
9. Stein S. & falck-Pedersen e., 2012. Sensing adenovirus infection: activation of interferon regulatory factor 3 in RAW 264.7 cells. J Virol. 86(8):4527-37.