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Blood, Vol. 111, Issue 3, 1021-1028, February 1, 2008
Transgenic blockade of interleukin 6 transsignaling abrogates inflammation
Blood Rabe et al.
111: 1021
Supplemental materials for: Rabe et al
Generation of sgp130Fc transgenic mice
Based on our previous generation of sIL6R transgenic mice using a PEPCK promoter,27 an expression construct was engineered containing the open reading frame for sgp130Fc inserted prior to a rat  -globin intron and a rat -globin polyA cassette (Figure S4A). Seven founder mice were obtained which differed in transgene copy number as determined by Southern blotting (Figure S4B and data not shown). The sgp130Fc serum levels of the four mice with the highest circulating sgp130Fc levels (between 60-500 ng/ml) are presented in Figure S4C. To test the inhibitory properties of the increased sgp130Fc levels displayed by these transgenic mice, the induction of the acute phase reactant, serum amyloid-A (SAA) was monitored in mice challenged with a chimeric IL6-sIL6R fusion protein, Hyper-IL6.23 As shown in Figure S4D, SAA expression in response to 400ng Hyper-IL6 was comparable in both sgp130Fc transgenic mice and wild type littermates suggesting that optimization of sgp130Fc expression was required.
Files in this Data Supplement:
- Table S1. Leukocyte recruitment in wild type, opt_sgp130Fc transgenic and IL6−/− mice (PDF, 86.1 KB)
- Figure S1. Optimization strategy of the sgp130Fc-cDNA (PDF, 462 KB)
- Figure S2. cDNA analysis of sgp130Fc and opt_sgp130Fc (PDF, 3.88 MB) -
(A) Histogram represents the overall codon frequency within the coding region; e.g. red bars indicate the frequency of extremely rare codons. Codon usage of the optimized genes: the most frequently used codons was set to 100 and the remaining were scaled accordingly (“relative adaptiveness”). (B) Plot represents the codon usage at the indicated codon positions. (C) Plot represents GC content at the indicated sequence positions.
- Figure S3. Alignment of sgp130Fc and opt_sgp130Fc (PDF, 2.50 MB) -
Identical bases are indicated with *. Altered bases are indicated in red. Overall homology is 78%.
- Figure S4. Characterization of transgenic mice expressing a non-optimized sgp130Fc cDNA (PDF, 241 KB) -
(A) sgp130Fc construct injected into fertilized mouse eggs (pTZ-pEPCK¬sgp130Fc-glob.intron). (B) Southern blot analysis. Genomic tail DNA from wild type (wt) and heterozygous (-/+) and homozygous (+/+) transgenic animals was digested with NcoI. The 32P-labeled Xba-cDNA fragment was used for hybridization. (C) Expression of sgp130Fc protein in sgp130Fc transgenic mice. Serum was prepared from 8-10-wk-old wild type mice and homozygous transgenic mice of the lines L1, L3, L4 and L6 and sgp130Fc protein level was determined by ELISA. Four animals per line were analyzed. Data represent mean values ± SD. (D) Induction of acute phase response by Hyper-IL6 in sgp130Fc transgenic and wild type mice. Northern blot analysis showing the liver expression of serum amyloid A (SAA2) 4 h after i.p. injection of 400 ng Hyper-IL6 per mice. A wild type animal (wt) injected with the same volume PBS served as a negative control. 5 µg total liver RNA were separated on a 1% formaldehyde containing agarose gel and probed with a 32P-labeled PCR fragment of the mouse SAA2 gene.
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