Department of Biochemistry & Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Department of Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.
Biotechniques. 2022 Mar;72(3):81-84. doi: 10.2144/btn-2021-0089. Epub 2022 Feb 4.
Acute myeloid leukemia patients with FMS-like tyrosine kinase 3-internal tandem duplications and mixed lineage leukemia-protein AF9 fusion proteins suffer from poor clinical outcomes. The MOLM-13 acute myeloid leukemia cell line harbors both of these abnormalities and is used in CRISPR experiments to identify disease drivers. However, experimental observations may be biased or inconclusive in the absence of experimentally validated positive control genes. We validated sgRNAs for knockdown of for cell proliferation and for knockdown and upregulation for cytarabine resistance control genes in MOLM-13 cells. We have provided a detailed CRISPR protocol applicable to both gene knockdown or activation experiments and downstream leukemic phenotype analyses. Inclusion of these controls in CRISPR experiments will enhance the capacity to identify novel myeloid leukemia drivers in MOLM-13 cells.
携带 FMS 样酪氨酸激酶 3 内部串联重复和混合谱系白血病蛋白 AF9 融合蛋白的急性髓系白血病患者临床预后较差。MOLM-13 急性髓系白血病细胞系同时存在这两种异常，并且用于 CRISPR 实验以鉴定疾病驱动基因。然而，如果没有经过实验验证的阳性对照基因，实验观察可能存在偏差或不明确。我们验证了针对 MOLM-13 细胞中细胞增殖的 sgRNA，以及针对 knockdown 和 upregulation 以控制阿糖胞苷耐药性的 sgRNA。我们提供了一个详细的 CRISPR 方案，适用于基因敲低或激活实验以及下游白血病表型分析。在 CRISPR 实验中包含这些对照将增强在 MOLM-13 细胞中鉴定新型髓样白血病驱动基因的能力。
