|Screen||In Vivo CpG Screen|
|Posted On||08/12/2010 10:02 AM|
|Author||Amanda L. Blasius|
|Science Writer||Nora G. Smart|
Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are more abundant in prokaryotic genomes than in eukaryotic genomes (1). CpG ODNs are internalized by immune cells and are specifically recognized by Toll-like receptor 9 (TLR9) present in the membranes of endosomes (2-4), leading to the activation of a MyD88-dependent, TRIF-independent pathway. Ultimately, stimulation of TLR9 leads to the production of multiple cyotokines and chemokines including type I IFNs (IFN-α/β). Multiple pathogens, including DNA viruses, are detected by the immune system through TLR9.
Experimental stimulation of TLR9 often utilizes phosphorothioate (PS)-ODN, in which one of the backbone oxygens is replaced by sulfur, rendering these ODN more nuclease-resistant than “natural” ODN with a phosphodiester (PD) backbone. These ODN make effective artificial ligands and can be divided into Class A (also known as D-type), ClassB (also known as K-type), and Class C ODNs; each with distinct immunological effects (5). CpG-A and CpG-C ODNs are strong inducers of type I interferons (6), particularly in plasmacytoid dendritic cells (pDCs),whereas CpG-B ODNs are weak inducers of IFN-αβ (5;7;8), but they induce DCs to mature and produce the tumor necrosis factor (TNF)-α cytokine (5;7).
As plasmacytoid dendritic cells (pDCs) are the only cell that produces significant amounts of type I IFNs in response to nucleic acids (TLR9 and TLR7 stimulation), the production of type I IFN in response to in vivo CpG-A injection is intended to identify mutations that would affect TLR9 signaling and type I IFN production in pDCs. Pathways probed by the CpG screen would include those involved in TLR signaling, pDC development and function, and type I IFN signaling as the production of large amounts of type I IFN from pDCs depends on a type I IFN receptor-mediated autocrine feedback loop (9).
CpG-A injection into mice will also be used to find mutants that are sensitized to immunological or other stresses. Stimulation of the immune system by CpG-A and other TLR ligands often causes adverse effects, such as fever and shock, due to the high production of cytokines and chemokines. Mutant mice that are sensitized to these stresses may succumb to low doses of injected CpG-A, which normally does not cause death in wild type animals. For more details, please see the Low Dose LPS Screen.
|Reagents and Solutions|
CpG 2216 (this is a CpG-A, in contrast to CpG-B used in the TLR Signaling Screen)
* = phosphorothioaction
DOTAP liposomal transfection reagent. Once opened, entire vial should be used on the same day (Roche Biosciences; Catalogue No. 11 811 177 001).
5mL polystyrene round bottom tubes
BD insulin syringe (Becton-Dickinson; Catalogue No. 329424)
L-929 cells (ATCC #CCL-1)
Dulbecco’s modified eagle medium
10% (v/v) heat-inactivated fetal bovine serum
200 IU/mL penicillin
200 mg/mL streptomycin
IFNβ standards (eBioscience, San Diego, CA; Catalogue No. 14-8311-63)
Reporter Lysis Buffer (Promega, Madison, WI; Catalogue No. E3971)
Luciferase Assay Reagent (Promega; Catalogue No. E1483)
Preparation of CpG for injection
In vivo CpG screening
Type 1 IFN bioassay
Table 1. Type 1 interferon ELISA plate layout.
Generation of CpG mutant stocks
|Critical Parameters and Troubleshooting|
CpG injection mixture should not sit around for very long, and should be used 20-30 minutes after CpG and DOTAP are mixed together. To ensure mix is used in a timely manner, make mix for only 10-20 mice at a time depending on experience with tail vein injections.
1. Cardon, L. R., Burge, C., Clayton, D. A., and Karlin, S. (1994) Pervasive CpG suppression in animal mitochondrial genomes, Proc. Natl. Acad. Sci. U. S. A 91, 3799-3803.
2. Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K. A., Monks, B. G., Knetter, C. F., Lien, E., Nilsen, N. J., Espevik, T., and Golenbock, D. T. (2004) TLR9 signals after translocating from the ER to CpG DNA in the lysosome, Nat. Immunol. 5, 190-198.
3. Leifer, C. A., Kennedy, M. N., Mazzoni, A., Lee, C., Kruhlak, M. J., and Segal, D. M. (2004) TLR9 is localized in the endoplasmic reticulum prior to stimulation, J. Immunol. 173, 1179-1183.
4. Ishii, K. J., Takeshita, F., Gursel, I., Gursel, M., Conover, J., Nussenzweig, A., and Klinman, D. M. (2002) Potential role of phosphatidylinositol 3 kinase, rather than DNA-dependent protein kinase, in CpG DNA-induced immune activation, J. Exp. Med. 196, 269-274.
5. Klinman, D. M. (2004) Immunotherapeutic uses of CpG oligodeoxynucleotides, Nat. Rev. Immunol. 4, 249-258.
6. Krug, A., Rothenfusser, S., Hornung, V., Jahrsdorfer, B., Blackwell, S., Ballas, Z. K., Endres, S., Krieg, A. M., and Hartmann, G. (2001) Identification of CpG Oligonucleotide Sequences with High Induction of IFN-alpha/beta in Plasmacytoid Dendritic Cells. Eur. J. Immunol. 31, 2154-2163.
7. Hemmi, H., Kaisho, T., Takeda, K., and Akira, S. (2003) The Roles of Toll-Like Receptor 9, MyD88, and DNA-Dependent Protein Kinase Catalytic Subunit in the Effects of Two Distinct CpG DNAs on Dendritic Cell Subsets. J. Immunol. 170, 3059-3064.
8. Vollmer, J., Weeratna, R., Payette, P., Jurk, M., Schetter, C., Laucht, M., Wader, T., Tluk, S., Liu, M., Davis, H. L., and Krieg, A. M. (2004) Characterization of Three CpG Oligodeoxynucleotide Classes with Distinct Immunostimulatory Activities. Eur. J. Immunol. 34, 251-262.