To investigate whether co-expression of PBX3/MEIS1 can mimic that of MLL-AF9, HOXA9/MEIS1 or HOXA9/PBX3 in inducing leukemogenesis, we conducted in vivo mouse bone marrow transplantation (BMT) assays.
More...To investigate whether co-expression of PBX3/MEIS1 can mimic that of MLL-AF9, HOXA9/MEIS1 or HOXA9/PBX3 in inducing leukemogenesis, we conducted in vivo mouse bone marrow transplantation (BMT) assays. Briefly, normal mouse bone marrow (BM) progenitor (i.e., lineage negative; Lin-) cells collected from B6.SJL (CD45.1) donor mice (CD45.1) were retrovirally co-transduced with MSCVneo-MLL-AF9+MSCV-PIG (MLL-AF9), MSCVneo-HOXA9+MSCV-PIG (HOXA9), MSCVneo-HOXA9+MSCV-PIG-MEIS1 (HOXA9+MEIS1), MSCVneo-HOXA9+MSCV-PIG-PBX3 (HOXA9+PBX3), MSCV-PIG-PBX3+MSCVneo-MEIS1 (PBX3+MEIS1), MSCVneo+MSCV-PIG-PBX3 (PBX3) , MSCVneo+MSCV-PIG-MEIS1 (MEIS1), or MSCVneo+MSCV-PIG (normal control; NC). Retrovirally transduced cells then were cultured with cytokines as well as puromycin and G418. Seven days later, the donor cells were transplanted into lethally irradiated (960 rads) 8- to 10-week-old C57BL/6 (CD45.2) recipient mice. The transplanted mice were watched for leukemogenesis. Then, gene expression profiling was conducted with bone marrow samples collected from leukemia groups and control group.
Overall design: A total of 20 mouse BM samples including 15 mouse AML samples with 3 each of the MLL-AF9, HOXA9+MEIS1, HOXA9+PBX3, HOXA9, and PBX3+MEIS1 groups, along with 5 samples from the normal control (NC) group, were analyzed by use of Affymetrix GeneChip Mouse Gene 2.0 ST Array (Affymetirx, Santa Clara, CA). For each sample, the CD45.1+ cells (i.e., transplanted donor cells) were sorted with flow cytometry from whole BM cells collected from BMT recipient mice at the end stage. Then total RNA was isolated by use of miRNeasy extraction kit (Qiagen, Valencia, CA).
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