All animal protocols were performed in accordance with the institutional animal care guidelines and conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication, 2011). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the University Of Louisville School Of Medicine. C3H/HeJ (Tlr4Lps-d, Stock no.: 000659) and C3H/HeOuJ (Stock no.:000635) mice aged 10–12 weeks were purchased from Jackson Laboratory (Bar Harbor, ME). The C3H/HeJ mice are endotoxin resistant due to a spontaneous mutation at the lipopolysaccharide response locus in the toll-like receptor 4 gene, Tlr4Lps-d, and we termed these mice as TLR4M hereafter. Whereas the C3H/HeOuJ is a substrain of C3H/HeJ, has normal TLR4, and we termed these mice as TLR4N hereafter. The animals were fed standard chow and regular water ad libitum.
C3h hej tlr4lps d
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The C3H/HeJ (Tlr4Lps-d) is a mouse strain that carries a spontaneous mutation in the Toll-like receptor 4 (Tlr4) gene, resulting in a non-functional Tlr4 protein. This mutation renders the mice hyporesponsive to lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria.
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3 protocols using c3h hej tlr4lps d
1
TLR4 Mutant Mice Endotoxin Resistance
2
Mouse Model of Endotoxin-Induced Inflammation
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The protocol was approved by the Institutional Animal Care and Utilization Committee of the University of California, Irvine. Eight- to 10-week-old male BALB/cJ, C3H/HeJ (Tlr4Lps-d), and C3H/HeOuJ mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). Mice were housed in isolator cages, kept on a 12-hour light–dark cycle, and provided with autoclaved bedding, water, and food. Solutions in volumes of 250 µL were injected intraperitoneally. During terminal anesthesia, blood, spleens, and lungs were obtained. Spleens were weighed, and lungs fixed in 10% buffered formalin were processed for histopathology at the Comparative Pathology Laboratory at the University of California, Davis. Plasma samples were subjected to bead-based immunoassays at Myriad RBM (Austin, Texas) for the 59 analytes of the RodentMAP version 2.0 panel. Quantitative reverse transcription polymerase chain reaction for the mouse heme oxygenase-1 gene (HMOX1) and β-actin transcripts was carried out as described on total RNA extracted from whole blood [7 (link)]. Standards were clones of the targets in a plasmid vector. HMOX1 messenger RNA (mRNA) copies were normalized per 1000 copies of β-actin mRNA.
3
NF-κB Reporter Mice for Research
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Transgenic reporter mice harboring a transgene consisting of NF-κB responsive elements upstream of a minimal promoter and a lacZ reporter fused in-frame at the 5′-end to a nuclear localization sequence (NF-κB-RE/NLS-lacZ)46 (link)47 (link)48 (link) was re-derived into and continuously maintained in the C57BL/6 inbred background in a specific-pathogen-free facility. Genotyping was performed with PCR using lacZ-specific primers and genomic DNA extracted from biopsied tails. Wild-type mice in 129/SvEv in-bred background were purchased from Taconic (Germantown, NY). Toll-like receptor 4 (TLR4) mutant mice C3H/HeJ (Tlr4LPS-d) and TLR4-wild type control mice C3H/HeOuJ (Tlr4LPS-n) were purchased from the Jackson Laboratory (Bar Harbor, Maine). All experiments on animals were performed in accordance with federal and local regulations and after official approval from the Institutional Animal Care and Use Committee of New York University School of Medicine.
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