E-MEXP-85 - Transcription profiling of mouse ck-OBF-1 transgene
Released on 25 October 2006, last updated on 2 May 2014
The Lck-OBF-1 construct used to generate transgenic mice contains an N-terminally HA epitope-tagged human OBF-1 cDNA under the control of the murine proximal lck promoter (-3100 to +23 relative to the transcription start site) that was purified by electroelution. Transgenic mouse lines were obtained and bred in B6CF1 x C57BL/6 background. For the analysis, age- and sex-matched littermates were used. The mice were kept in a standard mouse facility. Mice were sacrificed, the thymi were taken, rinsed in PBS and immediately homogenized in TRIZOL reagent (Life Technologies). Total RNA was prepared using TRIZOL reagent and purified on RNeasy Miniprep columns (Qiagen) according to the manufacturers' instructions. Array set (A1): Equal amounts of RNA from four young adult male mice (6.5 weeks old) per genotype were purified as stated above and pooled in equal amounts. Array set (A2): Equal amounts of total RNA from four young adult mice (6.5 weeks old; 2 males and 2 females each time) per genotype were purified as stated above and pooled in equal amounts. Both the WT and the transgenic pool were hybridized to Affymetrix GeneChips in duplicate. Array set (B): RNA was prepared from sorted thymic cell populations (CD4+ CD8+ CD25- and CD4+ CD8+ CD25+) from a pool of 15 young adult (mixed gender, 6 to 9 weeks of age) transgenic mice as stated above. Cell sorting was performed on a MoFlo (DakoCytomation) with a purity of at least 94%.
transcription profiling by array, stimulus or stress
Edward J Oakeley <firstname.lastname@example.org>, Boris A Bartholdy, Herbert Angliker, Patrick Matthias
The Ets factor Spi-B is a direct critical target of the coactivator OBF-1. Bartholdy B, Du Roure C, Bordon A, Emslie D, Corcoran LM, Matthias P.