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. 1999 Feb 15;189(4):615–625. doi: 10.1084/jem.189.4.615

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Tlr4 mutation analysis in C3H/HeJ and C57BL/10ScCr. (A) C3H/HeJ Lps mutation. Sequence analysis of Tlr4 shows the missense mutation, a C to A transversion (Pro⁷¹²His). This change is present only in C3H/HeJ mice. A silent mutation (C to T substitution, Asn⁷¹⁹) is also present in several inbred strains of mice, including A/J and BALB/cJ. (B and C) C57BL/10ScCr Lps mutation. (B) RT-PCR of spleen mRNA from C57BL/6J (lanes 3, 6, 9, 12, 17, 20, 23, and 26), C3H/HeJ (lanes 4, 7, 10, 13, 18, 21, 24, and 27), and C57BL/10ScCr (lanes 5, 8, 11, 14, 19, 22, 25, and 28) using six different combinations of primers. Molecular size markers λ DNA-HindIII digest (lanes 1 and 15) and φX-174 RF HincII digest (lanes 2 and 16) are shown. Primer positions are given in brackets according to the position of the first coding ATG. Lanes 3–5: fragment A (696 bp) was amplified with primers 5′-TCTGCGCTGCCACCAGTTAC-3′ (−69) and 5′-ACATGTCTAAAGAGAGATTGAC-3′ (627). Lanes 6–8: fragment B (1,200 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-GAAAGAATTGCCAGCCATTTT-3′ (1,442). Lanes 9 and 10: fragment C (2,700 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 17–19: fragment D (409 bp) was amplified with primers 5′-TGACACCCTCCATAGACTTC-3′ (1,541) and 5′-GGTATATCAGAAATGCTACA-3′ (1,950). Lanes 20–22: fragment E (641 bp) was amplified with primers 5′-GTCCTTGAGAAGGTTGAGAAGTCCC-3′ (2,301) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 23–25: fragment F (299 bp) was amplified with primers 5′-GAAGGAAGTAGCACTGACACCTTC-3′ (2,643) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 12–14 and 26–28: Gapdh (459 bp) was amplified with primers 5′-TTTGTGATGGGTGTGAACCACGAG-3′ and 5′-GGAGACAACCTGGTCCTCAGTGTA-3′. A single Tlr4 product of the expected size is seen only in RT-PCR of spleen mRNA from C57BL/6J and C3H/HeJ, although Gapdh mRNA was amplified from all RT-PCR samples including C57BL/10ScCr. (C) Southern blot analysis of chromosomal rearrangement associated with the C57BL/10ScCr Lps allele. Southern blot of a Tlr4 partial cDNA (position 242–1442) hybridized to C57BL/6J (lanes 1 and 5), C57BL/10ScCr (lanes 2 and 6), C3H/HeJ (lanes 3 and 7), and DBA/2J (lanes 4 and 8) DNA digested with BamHI (1– 4) or HindIII (5–8). Size markers (in kb) are indicated to the left of the autoradiogram.

Tlr4 mutation analysis in C3H/HeJ and C57BL/10ScCr. (A) C3H/HeJ Lps mutation. Sequence analysis of Tlr4 shows the missense mutation, a C to A transversion (Pro⁷¹²His). This change is present only in C3H/HeJ mice. A silent mutation (C to T substitution, Asn⁷¹⁹) is also present in several inbred strains of mice, including A/J and BALB/cJ. (B and C) C57BL/10ScCr Lps mutation. (B) RT-PCR of spleen mRNA from C57BL/6J (lanes 3, 6, 9, 12, 17, 20, 23, and 26), C3H/HeJ (lanes 4, 7, 10, 13, 18, 21, 24, and 27), and C57BL/10ScCr (lanes 5, 8, 11, 14, 19, 22, 25, and 28) using six different combinations of primers. Molecular size markers λ DNA-HindIII digest (lanes 1 and 15) and φX-174 RF HincII digest (lanes 2 and 16) are shown. Primer positions are given in brackets according to the position of the first coding ATG. Lanes 3–5: fragment A (696 bp) was amplified with primers 5′-TCTGCGCTGCCACCAGTTAC-3′ (−69) and 5′-ACATGTCTAAAGAGAGATTGAC-3′ (627). Lanes 6–8: fragment B (1,200 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-GAAAGAATTGCCAGCCATTTT-3′ (1,442). Lanes 9 and 10: fragment C (2,700 bp) was amplified with primers 5′-GCTGGATTTATCCAGGTG-3′ (242) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 17–19: fragment D (409 bp) was amplified with primers 5′-TGACACCCTCCATAGACTTC-3′ (1,541) and 5′-GGTATATCAGAAATGCTACA-3′ (1,950). Lanes 20–22: fragment E (641 bp) was amplified with primers 5′-GTCCTTGAGAAGGTTGAGAAGTCCC-3′ (2,301) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 23–25: fragment F (299 bp) was amplified with primers 5′-GAAGGAAGTAGCACTGACACCTTC-3′ (2,643) and 5′-AAATTGGAATGAAGACCTCTCA-3′ (2,942). Lanes 12–14 and 26–28: Gapdh (459 bp) was amplified with primers 5′-TTTGTGATGGGTGTGAACCACGAG-3′ and 5′-GGAGACAACCTGGTCCTCAGTGTA-3′. A single Tlr4 product of the expected size is seen only in RT-PCR of spleen mRNA from C57BL/6J and C3H/HeJ, although Gapdh mRNA was amplified from all RT-PCR samples including C57BL/10ScCr. (C) Southern blot analysis of chromosomal rearrangement associated with the C57BL/10ScCr Lps allele. Southern blot of a Tlr4 partial cDNA (position 242–1442) hybridized to C57BL/6J (lanes 1 and 5), C57BL/10ScCr (lanes 2 and 6), C3H/HeJ (lanes 3 and 7), and DBA/2J (lanes 4 and 8) DNA digested with BamHI (1– 4) or HindIII (5–8). Size markers (in kb) are indicated to the left of the autoradiogram.