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Blood, Vol. 110, Issue 7, 2685-2695, October 1, 2007
A2A adenosine receptors and C/EBPß are crucially required for IL-10 production by macrophages exposed to Escherichia coli
Blood Csóka et al.
110: 2685
Supplemental materials for Csoka et al, Vol. 110, Issue 7, 2685-2695
Files in this Data Supplement:
- Figure S1. Generation of A2B receptor KO mice (JPG, 62.2 KB)
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(A) A2B KO mice were generated by the insertion of an internal ribosome entry site (IRES)-lacZ-Neo cassette into exon 1 of the wild type A2B allele using standard homologous recombination techniques in embryonic stem cells. Genotyping was conducted on genomic DNA isolated from tails using endogenous A2B primers 1, 5′-GTACGTGGCGCTGGAGCTGGTTATC-3′ and 2, 5′-GCTCTGTGTGAGCACCAGCACGAAG-3′ and the neomycin primer 3, 5′-GGGTGGGATTAGATAAATGCCTGCTCT-3′. A combination of primers 1 and 2 give an amblicon of 247 bps corresponding to the wild type A2B allele, while primers 2 and 3 yield a 417 bp amplicon corresponding to the mutant A2B allele. (B) Total cellular RNA was isolated from the large intestine and bladder of A2B receptor wild type (+/+), heterozygous (+/−) and KO (−/−) mice and real time RT-PCR was used to quantify A2B receptor transcripts. Methods for RNA isolation and primers and PCR conditions can be found in Sun et al, 2005.1 Results demonstrated half normal A2B receptor transcripts in heterozygous tissues and the absence of A2B receptor transcripts in KO tissues, verifying the presence of a null A2B allele in these animals.
1Sun C-X, Young HW, Molina JG, Volmer JB, Schnermann J, Blackburn MR. A protective role for the A1 adenosine receptor in adenosine-dependent pulmonary injury. J Clin Invest. 2005;115:35-43.
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