O papel modulador do gene AIRE - capes

225Table 1. Target cDNA sequences amplified by RT-PCR, oligonucleotide primers and probes aTarget Forward primer Reverse primer Internal probeIL-1α TTACAGTGAAAACGAAGA TGTTTGTCCACATCCTG GAGAACCTCTGAAACGTCIL-1β GCAACTGTTCCTGAACTC CTCGGAGCCTGTAGTGCA ATTGTGGCTGTGGAGAAIL-2 AACAGCGCACCCACTTCAA TTGAGATGATGCTTTGACA GTTCATCTTCTAGGCACTGIL-3 GCCAGCTCTACCACCAGCA AACATTCCACGGTTCCACG AGGTTCTGGGAGCTTCCCIL-4 TAGTTGTCATCCTGCTCTT CTACGAGTAATCCATTTGC GATGATCTCTCTCAAGTGIL-6 TTCCTCTCTGCAAGAGACT TGTATCTCTCTGAAGGACT CATTTCCACGATTTCCCAIL-7 ACATCATCTGAGTGCCACA CTCTCAGTAGTCTCTTTAG TGCCTTGTGATACTGTTAGTNF-β TCAGAAGCACTTGACCCAT AAGTCCCGGATACACAGACT CAAAGTAGAGGCCACTGGIF-γ AACGCTACACACTGCATCT TGCTCATTGTAATGCTTGG TCGCCTTGCTGTTGCTGTGM-CSF TTCCTGGGCATTGTGGTCT TGGATTCAGAGCTGGCCTGG ACCTCTTCATTCAACGTGIL2-Rβ CCAGCTGCTTCACCAACCA AGAAGAGCAGCAGGTCATC CCTGCCAGGTGTACTTRAG-1 ATGGCTGCCTCCTTGCCGTCT GTATCTCCGGCTGTGCCCGTC – – – – – – – – – – – – – –RAG-2 ATGTCCCTGCAGATGGTAACA TAAATCTTATCGGAAAGCTCA – – – – – – – – – – – – – –MHC-I GGGGATGGAACCTTCCAGAAGTG CACAAAAGCCACCACAGCTCCA – – – – – – – – – – – – – –MHC-II CGACTGTAGAACCTTAGCCTG GTGGACACAATTCCTGTTTT – – – – – – – – – – – – – –MMTV GTAGACCTAATTGTATTTATAAATGTT GGACTTATAGGGGACCTTACAT TGCTATAGCTCCCTCCCTCTTCGβ-ACTIN ATGGATGACGATATCGCT ATGAGGTAGTCTGTCAGGT AGCAAGAGAGGTATCCTaProbes for cytokines, β-actin and MHC-I and MHC –II see ref. [26]; for RAG-1 and RAG-2 see [27] and for MMTV see [28].Hierarchical clusteringA)The gene expression data analyzed here were collected, fromthree independent determinations, on blotted RT-PCR of severalgene products from different developmental stages of thethymus of Balb-c, C57Bl/6 and CBA/J strains. We used ahierarchical clustering algorithm, comparing means of differentgenes whose standard deviation do not overlaps and,whose objective, is to compute a dendrogram that assemblesall elements into a single tree. The software for this algorithmcan be obtained from the author http://rana.stanford.edu/clustering[13, 14].Balb-cCBA/JC57BL/614Postcoitum development15 16 17 NB AD▲ResultsQuantification of TCRVβ8.1 rearrangement in developingthymusWe detected temporal and quantitative differences in the onsetof V(D)J recombination of TCRVβ8.1-Dβ2.1 DNA segmentsamong the strains studied.The Balb-c strain started recombination at 16–17 days p.c.,CBA/J at 16 days p.c and C57Bl/6 at 14–15 days p.c. (Fig. 1).The CBA/J strain drastically reduced the rearranged DNAsegment at 17 days p.c., reappearing in newborn and adultthymus.Waves of gene expressionThe developing thymus of the three strains displayed threegene clusters according to their waves of expression levelsFig. 1. V(D)J recombination between TCRVβ8.1 and Dβ2.1 during thymusdevelopment investigated by PCR. (A) Southern-blot of PCR products.(B) Quantification of hybridizations using a phosphor-imager. Arbitrary valuesexpressed in DLU (digital light units) as displayed by the OptiQuant ®software.