Protocols


Screen (pdf version)
ScreenLPS Hypersensitivity Screen
Posted On02/18/2010 12:24 PM
AuthorAmanda L. Blasius, Sungyong Won
Science WriterNora G. Smart
Background
LPS or lipopolysaccharide is a glycolipid of the outer membrane of Gram-negative bacteria, and is a ligand for toll-like receptor 4 (TLR4) (1).  LPS has long been known to cause fever, shock, and sometimes death by inducing cytokine production, particular tumor necrosis factor (TNF), by hematopoietic cells (2-4).  However, recognition of LPS by cells of the innate immune system permits effective clearance of a Gram-negative infection (5).  Intraperitoneal (i.p.) injection of mice with a high enough dose of LPS will cause death in wild type C57BL/6J animals, with 100% death at 300μg/20g bodyweight (Table 1) (6).  Animals with mutations in the TLR4 signaling pathway or mutations that prevent the production of inflammatory cytokines are resistant to LPS-induced shock (1;7-9)
 
The low dose LPS screen was devised after the identification of an N-ethyl-N-nitrosourea (ENU)–induced mutation that caused mouse cytomegalovirus (MCMV) susceptibility (MCMV Susceptibility and Resistance Screen), termed mayday.  These mice succumbed to normally survivable doses of MCMV infection.  Moreover, they were generally more susceptible to spontaneous death, particularly when under added stresses such as viral infection (6)Mayday mice were found to have a mutation in Kcnj8, which encoded for a subunit of an ATP-sensitive potassium channel required for cardiac function (10).  In addition to MCMV susceptibility and spontaneous death, homozygous mayday mice were also sensitive to injections of low dose LPS (Table 1), with 100% dying at a dose of 50μg/20g bodyweight.  These mutants also succumb to i.p. doses of CpG containing oligonucleotides (127μg/20g bodyweight), which are recognized by TLR9, and double-stranded RNA or poly I:C (20μg/20g bodyweight), which are TLR3 ligands (6)
 
Table 1. Mortality in mayday homozygotes after LPS administration.
 
Dose (μg per mouse, IP) LPS
Wild-type (dead/injected)
Mayday (dead/injected)
0.01
0/5
2/21
0.05
0/5
8/20
0.1
0/5
10/20
1.0
0/5
7/20
5.0
0/5
25/30
10
0/5
18/26
50
0/4
5/5
100
0/4
ND
200
6/10
ND
300
10/10
ND
 
Based on the susceptibility of mayday homozygous mice to LPS, a screen for additional mutants that may sensitize mice to immunological or other stresses was devised.  ENU-mutagenized G3 mice are injected with a low dose of LPS based on the doses reported in Table 1 (5-10μg/mouse).  Alternatively, such mutations may also be identified by intravenous (i.v.) injected CpG-induced death (please see the In Vivo CpG Screen). The ability of the low dose LPS screen to pick up appropriate mutants was confirmed with the identification of a new allele of Kcnj8, termed sos.
 
An additional screen now in use uses ENU-mutagenized mayday mice to identify new mutations that can rescue the original hypersensitivity to LPS conferred by the mayday mutation.  ENU-mutagenized homozygous G3 mayday mice are injected with a low dose of LPS (10μg/mouse; I.V. injection).  Survivors of this initial low dose are then retested with 20μg/mouse of I.V. injected LPS.  
 
Reagents and Solutions
LPS (Alexis, San Diego, CA; Catalogue No. 581-008-L002)
 
Sterile PBS
 
BD insulin syringe (Becton-Dickinson; Catalogue No. 329424)
Method
Preparation of LPS
  1. Dilute LPS in sterile PBS to 10μg in 200μl.
  2. For retesting, dilute LPS in sterile PBS to 20μg LPS in 200μl..
In vivo LPS 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. G3 mice are injected with 10μg LPS i.p; 200μl per mouse.
  3. Check mice for death every day for five days (death usually occurs between 24-48 hours after injection).
  4. Generate homozygous stocks.  Please see the Genetic Mapping protocol. 
  5. Retest mice from the stock with 20μg LPS i.p. to confirm phenotype.
     
Alleles Identified
mayday
sos
References
   5. Rosenstreich, D. L., Weinblatt, A. C., and O'Brien, A. D. (1982) Genetic Control of Resistance to Infection in Mice. CRC Crit. Rev. Immunol. 3, 263-330.