|Coordinate||106,224,152 bp (GRCm38)|
|Base Change||T ⇒ A (forward strand)|
|Gene Name||toll-like receptor 9|
|Chromosomal Location||106,222,598-106,226,883 bp (+)|
|MGI Phenotype||Mice homozygous for a knock-out allele exhibit impaired immune system response to LPS, CpG, and Leishmania bazillensis infection.|
|Amino Acid Change||Valine changed to Glutamic Acid|
|Institutional Source||Beutler Lab|
V214E in : ENSMUSP00000082207 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||decrease in response to injected CpG DNA, immune system, MCMV susceptibility, TLR signaling defect: TNF production by macrophages, TLR signaling defect: type I IFN production by macrophages|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Local Stock||Live Mice, Sperm, gDNA|
|Last Updated||05/13/2016 3:09 PM by Peter Jurek|
The CpG3 phenotype was identified in a G3 screen for mutants with impaired response to Toll-like receptor (TLR) ligands (TLR Signaling Screen). Peritoneal macrophages from CpG3 mice produce normal amounts of tumor necrosis factor (TNF)-α in response to all TLR ligands tested, except oligodeoxynucleotides containing CpG motifs (CpG ODNs). In response to CpG ODN treatment, homozygous CpG3 macrophages produce no TNF-α. In addition, naïve B cells from whole blood of homozygous CpG3 mice fail to proliferate upon stimulation with CpG ODN in vitro, a response recently demonstrated to be specific for CpG ODN among other TLR agonists (1) (Figure 1).
Although not all of the same phenotypes have been examined, those tested are identical between CpG1, CpG2, CpG3 and CpG5 mice. (CpG3 heterozygote phenotypes have not been tested; CpG3 is tentatively classified as semidominant). Sequence analysis revealed that all four strains contain mutations in Tlr9. However, the positions of the mutations differ, with the CpG1, CpG3 and CpG5 mutations located in the sixteenth, sixth and fourteenth extracellular leucine-rich repeats (LRR), respectively, and the CpG2 mutation located in the cytoplasmic Toll/IL-1R (TIR) domain. In addition to CpG1, CpG2, CpG3 and CpG5, another strain of mice, designated effete, also exhibits impaired TNF-α responses to CpG ODN treatment. The mutant has no TLR9 mutation; the causative mutation is under investigation.
|Nature of Mutation|
The CpG3 mutation corresponds to a T to A transversion at position 747 of the Tlr9 transcript, in exon 2 of 2 total exons.
The mutated nucleotide is indicated in red lettering, and causes a valine to glutamic acid substitution at residue 214 of the TLR9 protein.
Please see the record for CpG1 for information about Tlr9.
The CpG3 mutation substitutes glutamic acid for valine at position 214 of the TLR9 protein, which lies in the predicted sixth LRR module of the TLR9 ectodomain and is conserved in human TLR9. No tertiary structural data presently exist for TLR9, making it difficult to hypothesize how the CpG3 mutation could affect either ligand binding or receptor dimerization. Recently, the crystal structure of the related TLR3 heterodimer bound to double-stranded (ds) RNA has been elucidated (see record for CpG1). The ligand-bound TLR3 heterodimer forms an M-shape with the dsRNA binding to the concave surfaces of the TLR3 heterodimer at two locations on each ectodomain (2). It has been hypothesized that TLR7, 8 and 9 ligands may also bind to the concave surface of the ectodomain at a site made up by insertions at LRR 2, 5, 8 and 11 (3). The CpG3 mutation might somehow disrupt ligand binding and/or receptor dimerization, or destroy proper folding or localization of the receptor. The CpG3 phenotype supports any and all of these possibilities.
|Primers||Primers cannot be located by automatic search.|
CpG3 genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide change.
Primers for PCR amplification
CpG3(F): 5’- GGACGGGAACTGCTACTACAAGAAC-3’
CpG3(R): 5’- ATTGTGTGCCAGGCTAAGGCTC-3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 7:00
7) 4°C ∞
Primers for sequencing
CpG3_seq(F): 5’- TGAGCAATCTCACCCATCTG -3’
CpG3_seq(R):5' - GCAAAGGATACCTTCTTGCG -3'
NOTE: The forward PCR primer works better for forward sequencing:
CpG3(F): 5’- GGACGGGAACTGCTACTACAAGAAC-3’
The following sequence of 1237 nucleotides (from Genbank genomic region NC_000075 for linear DNA sequence of Tlr9) is amplified:
1441 ggaactgcta ctacaagaac ccctgcacag gagcggtgaa ggtgacccca ggcgccctcc
1501 tgggcctgag caatctcacc catctgtctc tgaagtataa caacctcaca aaggtgcccc
1561 gccaactgcc ccccagcctg gagtacctcc tggtgtccta taacctcatt gtcaagctgg
1621 ggcctgaaga cctggccaat ctgacctccc ttcgagtact tgatgtgggt gggaattgcc
1681 gtcgctgtga ccatgccccc aatccctgta tagaatgtgg ccaaaagtcc ctccacctgc
1741 accctgagac cttccatcac ctgagccatc tggaaggcct ggtgctgaag gacagctctc
1801 tccatacact gaactcttcc tggttccaag gtctggtcaa cctctcggtg ctggacctaa
1861 gcgagaactt tctctatgaa agcatcaccc acaccaatgc ctttcagaac ctaacccgcc
1921 tgcgcaagct caacctgtcc ttcaattacc gcaagaaggt atcctttgcc cgcctccacc
1981 tggcaagttc ctttaagaac ctggtgtcac tgcaggagct gaacatgaac ggcatcttct
2041 tccgcttgct caacaagtac acgctcagat ggctggccga tctgcccaaa ctccacactc
2101 tgcatcttca aatgaacttc atcaaccagg cacagctcag catctttggt accttccgag
2161 cccttcgctt tgtggacttg tcagacaatc gcatcagtgg gccttcaacg ctgtcagaag
2221 ccacccctga agaggcagat gatgcagagc aggaggagct gttgtctgcg gatcctcacc
2281 cagctccgct gagcacccct gcttctaaga acttcatgga caggtgtaag aacttcaagt
2341 tcaccatgga cctgtctcgg aacaacctgg tgactatcaa gccagagatg tttgtcaatc
2401 tctcacgcct ccagtgtctt agcctgagcc acaactccat tgcacaggct gtcaatggct
2461 ctcagttcct gccgctgact aatctgcagg tgctggacct gtcccataac aaactggact
2521 tgtaccactg gaaatcgttc agtgagctac cacagttgca ggccctggac ctgagctaca
2581 acagccagcc ctttagcatg aagggtatag gccacaattt cagttttgtg acccatctgt
2641 ccatgctaca gagccttagc ctggcacaca at
PCR primer binding sites are underlined; sequencing primer binding sites are highlighted in gray; the mutated T is shown in red text.
1. Jiang, W., Lederman, M. M., Harding, C. V., Rodriguez, B., Mohner, R. J., and Sieg, S. F. (2007) TLR9 stimulation drives naive B cells to proliferate and to attain enhanced antigen presenting function, Eur. J Immunol. 37, 2205-2213.
2. Liu, L., Botos, I., Wang, Y., Leonard, J. N., Shiloach, J., Segal, D. M., and Davies, D. R. (2008) Structural basis of toll-like receptor 3 signaling with double-stranded RNA, Science 320, 379-381.
|Science Writers||Eva Marie Y. Moresco|
|Illustrators||Diantha La Vine|
|Authors||Michael Berger, Bruce Beutler|