O papel modulador do gene AIRE - capes

laboratory using a Generation III Array Spotter (Amersham-MolecularDynamics, Sunnyvale, CA, USA). The complete file providing all genes presentin the microarrays used in this study is available online at(http://rge.fmrp.usp.br/passos/nylon_array/mtb1).Mouse expressed sequence tag (EST) cDNA clones were obtained from theSoares thymus 2NbMT normalized library, prepared from a C57Bl/6J 4-weekoldmale thymus, and available at the I.M.A.G.E. Consortium(http://image.llnl.gov/image/html/iresources.shtml).cDNA inserts were homogeneous in size (approximately 1 Kb) and cloned inthree vectors (pT7T3D, pBluescript and Lafmid) and were amplified in 384- or96-well plates using vector-PCR amplification with the following primers, whichrecognize the three vectors, LBP 1S 5’-GTGGAATTGTGAGCGGATACC-3’forward and LBP 1AS 5’-GCAAGGCGATTAAGTTTGG-3’ reverse.The membranes were first hybridized with the LBP 1AS[γ- 33 P]dCTP (GEHealthcare, USA) labeled oligonucleotide (vector hybridization). Quantificationof the signals obtained permitted the estimation of the amount of DNAdeposited in each spot. After stripping, the membranes were subsequently usedfor hybridization with thymus cDNA complex probes (sample hybridization).The annotation of each cDNA sequence was updated using S.O.U.R.C.E.(http://genome-www5.stanford.edu/cgi-bin/source/sourceSearch) and DAVID(http://david.abcc.ncifcrf.gov) databases, allowing for a set of information: genename, sequence, biological and molecular functions.241