Sox17-transgenic hematopoietic stem cell microarray

The transcription factor SOX17 is expressed by fetal, but not adult hematoipoietic stem cells (HSCs), and is required for the maintenance of fetal and neonatal, but not adult, HSCs. In the current study we show that ectopic expression of Sox17 in adult HSCs and transiently reconstituting multipotent progenitors was sufficient to confer increased self-renewal potential and the expression of fetal HSC genes including fetal HSC surface markers. To assess the acute effects of ectopic Sox17 expression on global gene expression in adult HSCs, we performed microarray analysis to compare the gene expression profile of adult Sox17-trangenic and control HSCs after short induction of Sox17-transgene expression. Total RNA were isolated from 5 independent, freshly isolated aliquots of 10,000 HSCs isolated from 8-week old Sox17-transgenic ((tetO)7CMVSox17-IRES-NucEGFP;B6.Cg-Gt(ROSA)26Sortm1(rtTA*M2)Jae/J double transgenic) or littermate control mice that were treated with doxycycline for 5 days to induce transgene expression. Purified RNA was reverse transcribed and amplified using the WT-Ovation™ Pico RNA Amplification system (NuGEN Technologies) following the manufacturer’s instructions. Sense strand cDNA was generated using WT-Ovation™ Exon Module (NuGEN), then fragmented and labeled using the FL-Ovation™ cDNA Biotin Module V2 (NuGEN). 2.5µg of labeled cDNA were hybridized to Affymetrix Mouse Gene ST 1.0 microarrays.