Effect of HIF-1alpha knockout in gene expression profile of osteosarcoma cells in vivo.
To explore in vivo changes in gene expression of osteosarcoma cells upon depletion of HIF-1alpha we performed RNAseq analysis using Mock control AXT cells and HIF-1alpha KO AXT cells. HIF1alpha was knocked out using CRISPR-Cas9 with two different target sequences. KO1-1 and KO1-2 cells are derived from target sequence#1:5'- AGATGTGAGCTCACATTGTGGGG -3' (exon3) and KO2 cells are from  #2:5'-GCTAACAGATGACGGCGACATGG-3' (exon5). AXT cells were established from BMSCs of Ink4a/Arf null mouse by overexpressing human c-MYC (Shimizu T, et al. Oncogene, 2010). Overall design: 2x10^6 Mock control cells and HIF-1alpha KO cells emmbedded in matrigel were inoculated into C57BL/6scid mice and harvested tumors next day. Tumors were disrupted using a BioMasher (Nippi, Tokyo, Japan) and total RNA was extracted.