Molecular mechanisms in Murine Syngeneic Leukemia Stem Cells

Cancer treatments usually gain good response, but some tumors relapse frequently. Acute My-eloid Leukemia (AML) is notorious for its robust relapse. This is attributed to the Leukemic Stem Cells (LSCs). We use a murine syngeneic leukemia model, ML23, in search for possible identifi-cation and study of LSCs in syngeneic settings. Hereby, we present prospective isolation of a de-fined LSC sub-population, encompassing the potency to passage disease from mouse to mouse. We further provide molecular insights of whole transcriptome analysis. Importantly, the ML23 LSC sub-population is expressing therapeutic targeted genes and provides a model for research in immune-competent animals. ML23 leukemia line was generated as previously reported [Keinan et al; Cell Death & Disease (2021) 12: 193]. essentially, transgenic rtTA mice (Jax strein 6965) cells transformed using lentiviruses over expressing oncogenic factors. ML23 cells were passaged in C57BL/6 mice. Seq description: We used the NEXTflex Rapid Directional RNA-Seq Kit (catalog # NOVA-5138-01) for li-brary construction; we then used the Qubit DS DNA chip for quality assurance of the samples. The samples were then sent to the G-INCPM facility for RNA-sequencing. Cells were frozen in SMARTer buffer (100 cells per sample, in 10.5 µL) and stored at -80° C. Li-brary preparation and sequencing were carried out at the INCPM facility (Rehovot, Israel), yielding 5-15 million single-reads of 61 bases per sample. Data were analyzed using Partek (http://www.partek.com) online services, briefly: alignment carried out with STAR v2.5.3a (reference index: mm10 - Ensembl Transcripts release 92), quantification with the in-house Partek algorithm (Quantify to annotation model (Partek E/M)) followed by nor-malization and differential expression analysis DEseq2 v3.5. Annotated GO gene lists were obtained from MGI (http://www.informatics.jax.org/function.shtml).