O papel modulador do gene AIRE - capes

Among the genetic nodes observed, those implicated in intracellular proteintransport were the most interactive, which may represent important geneticcascades during the development of the thymus/thymocytes.Material and methodsFetal thymus and RNA preparationThe (♀Balb-c x ♂C57Bl/6) F 1 hybrid mice were obtained in our ownlaboratory. The animals were maintained in a 0.45µ air filtered isolator, on a 12hour light/dark cycle, a temperature of 23 o C ± 2 o C and feed and water adlibitum. Pregnant Balb-c females were sacrificed by cervical dislocation and thefetuses were surgically collected from the uterus. The age of the fetuses (indays post coitum, p.c.) was confirmed on the basis of the morphologicalcharacteristics of each development phase [23] (Rugh, 1968). Thymic samplescollected from 14 to 17 days p.c. were used, a stage when the maturation of thethymocytes and the emergence of the T cell receptor V beta genes occur [7,18].Fetal thymus tissue (1 mg containing approximately 1 x 10 7cells) wasobtained by surgery under a stereomicroscope and immediately processed fortotal RNA extraction using Trizol® reagent (Invitrogen). Only protein- andphenol-free RNA preparations were used, as determined by UVspectrophotometry, and those featuring native undegraded RNA species instandard denaturing agarose gel electrophoresis.cDNA microarray methodA pair of cDNA microarrays was used, containing a total of 1,576 clones inthe form of PCR products, spotted in duplicate on 2.5 x 7.5 cm Hybond N+nylon membranes (GE Healthcare). The arrays were prepared in our own240