GSE124304
Fubp1 knockdown in BCR-ABL1 induced chronic myeloid leukemia (CML) like myeloproliferative neoplasm mouse model
We examine the oncogenic function of the Far Upstream Element Binding Protein 1 (FUBP1) in leukemia-initiating cells. For this perpose we performed knockdown of Fubp1 in CML cells in our mouse model. Here, we transduce donor bone marrow cell with the oncogene BCR-ABL1 and Fubp1 or scrambled shRNA and transplanted in recipient mice. Lineage- BCR-ABL1+ shRNA+ cells were then sorted from diseased mice and analyzed by RNA sequencing. We compare the gene expression of the Fubp1 shRNA expressing CML cells versus the control (scrambled shRNA expressing) cells in order to identify the relevant Fubp1 target genes. Overall design: Mouse donor BM cells that were harvested from 5-fluorouracil-treated mice, cotransduced twice with cryopreserved and thawed retrovirus expressing MSCV-IRES-RFP BCR-ABL1 and pLKO.1-Venus Fubp1- or scrambled shRNA lentivirus. 2.5-3x10^5 transduced cells were transplanted intravenously into sublethally irradiated (900 cGy) recipient mice. Lineage- BCR-ABL1-RFP+ shRNA-Venus+ DAPI- cells from bone marrow of CML mice (4 mice in the Fubp1 and 4 mice in the scrambled shRNA group) were sorted directly into TRIzol. RNA isolation, library preparation and next generation sequencing were performed by Transcriptome and Genome Analysis Laboratory (TAL, Göttingen, Germany), as described previously.24, 25 RNA-Seq data will be deposited in the GEO database upon acceptance of this manuscript. Read counts per gene were statistically analysed in R (version 3.4.3) using 'RStudio' (version 1.1.423). Differential analysis was performed using the 'DESeq2' tool (version 1.18.1). For annotation of genes with mouse gene names and human orthologue HGNC symbols, the 'biomaRt' package (version 2.34.2) was used. Candidate genes were filtered by a padj value < 0.05 and a log2 fold change < -1 or >1 and visualised in a MA-plot. Principal component analysis was computed with the 'prcomp' function and results were visualized using the 'pca3d package'(version 0.10). Gene set enrichment analysis was performed using the 'GSEAPreranked' tool from the GSEA software (version 2.2.3; Broad Institute).