Protocols


Screen (pdf version)
ScreenIn Vivo CpG Screen
Posted On08/12/2010 10:02 AM
AuthorAmanda L. Blasius
Science WriterNora G. Smart
Background
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)
G*G*GGGACGATCGTCG*G*G*G*G*G
* = phosphorothioaction
 
DOTAP liposomal transfection reagent. Once opened, entire vial should be used on the same day (Roche Biosciences; Catalogue No. 11 811 177 001).
 
Sterile PBS
 
5mL polystyrene round bottom tubes
 
BD insulin syringe (Becton-Dickinson; Catalogue No. 329424)
 
L-929 cells (ATCC #CCL-1)
 
L-929 medium
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)
Method
Preparation of CpG for injection
  1. Add 2μg CpG to 100μL sterile PBS in a 5 mL polystyrene round bottom tube.
  2. Add 12μL DOTAP to 88μL sterile PBS in a 5 mL polystyrene round bottom tube.
  3. Incubate for 5 minutes at room temperature.
  4. Mix CpG and DOTAP solutions.
  5. Incubate 20 minutes at room temperature, mix again gently, inject into mice (see below).
In vivo CpG screening
  1. ENU-mutagenized G0 C57BL/6 mice are generated and G3 mice generated.  Please see the Genetic Mapping protocol for more details and breeding schemes. 
  2. Inject G3 mice into the tail vein with 200μL CpG/DOTAP solution.
  3. Cages are time stamped at the time of injection.
  4. Bleed mice after 6 hours. 
  5. Collect serum by spinning blood down at 1000 rpm for 5 minutes and collecting the supernatant.
  6. Test serum in type I IFN bioassay (see below)
Type 1 IFN bioassay
  1. On the day prior to bioassay, 5x104 L-929-ISRE cells are added to every well of a white, tissue culture-treated 96 well flat-bottomed plate in a volume of 50μL L-929 medium, and incubated overnight at 37oC/5%CO2.
  2. A mouse recombinant IFNβ standard series is prepared immediately prior to bioassay, using 7 serial two-fold dilutions (starting from ~200U/µL for a final concentration of 100U/μL) and L-929 medium alone.
  3. Dilute serum samples 1:10 for a final concentration of 1:20 using L-929 medium.
  4. 50μL of each standard point or sample is added to the L-929-ISRE plate as organized in Table 1.
Table 1. Type 1 interferon ELISA plate layout.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
IFNβ
 
 
1
2
3
4
5
6
7
8
9
10
11
12
 
A
sample 1
sample 2
sample 3
sample 3
sample 4
sample 5
sample 6
sample
7
sample
8
sample
9
sample
10
100
 
B
etc.
 
 
 
 
 
 
 
 
 
50
 
C
 
 
 
 
 
 
 
 
 
 
 
25
 
D
 
 
 
 
 
 
 
 
 
 
 
12.5
 
E
 
 
 
 
 
 
 
 
 
 
 
6.25
 
F
 
 
 
 
 
 
 
 
 
 
 
3.125
 
G
 
 
 
 
 
 
 
 
 
 
 
1.6125
 
H
 
 
 
 
 
 
 
 
 
 
 
0
 
  1. Incubate at 37oC/5%CO2 for 6 hours.
  2. Following incubation, supernatant is discarded and plates are washed once with 200μL/well PBS.
  3. PBS is then discarded, and 30μL/well reporter lysis buffer is added.
  4. Plates are incubated at -70oC for greater than one hour.
  5. After plates have thawed, 50μL/well of Luciferase Assay Reagent is added, and luminescence read immediately.
     
    Bioactive type 1 IFN concentrations are inferred from the standard curve of [IFNb] versus ISRE-dependent luminescence.
Generation of CpG mutant stocks
  1. Generate homozygous stocks.  Please see the Genetic Mapping protocol. 
  2. Repeat CpG screen on mice to confirm phenotype.
     
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.

Alleles Identified
2018
bullet_gray
cellophane
Cpg1
Cpg3
Cpg6
domino
earl_grey
feeble
Gemini
inept
Meager
otiose
pam_gray
pococurante
References