Cytokine Stimulation of ADAR-deleted B16 murine melanoma cells

ADAR was deleted from the B16 murine melanoma line using CRISPR/Cas9. The loss-of-function phenotype was assessed in vitro with cytokine stimulation or vehicle control. Overall design: In order to determine the effect of an interferon sensitivity phenotype caused by ADAR deletion, B16 murine melanoma cancer cells were transiently transfected with plasmids encording Cas9 and guide RNA (sgRNA) against Adar or a non-targeting sequence. Cells were then subcloned and assessed for Adar deletion. Once verified, the Adar-null cells were treated with vehicle control (PBS with 0.1% BSA), interferon-beta (IFNb), or interferon-gamma (IFNg), and transcriptionally profiled by directional, paired-end RNAseq after a 36 hour stimulation.

研究人员通过CRISPR/Cas9技术，将B16小鼠黑色素瘤细胞系中的ADAR基因敲除，并借助体外细胞因子刺激或溶剂对照实验，对其功能丧失表型进行评估。
整体实验设计：为明确ADAR缺失所介导的干扰素敏感性表型效应，研究人员将靶向Adar的编码Cas9与单引导RNA（single guide RNA，sgRNA）的质粒，以及非靶向序列对照质粒，瞬时转染至B16小鼠黑色素瘤细胞中。随后对转染后的细胞进行亚克隆，并验证Adar基因的敲除效果。经验证确认ADAR基因缺失成功的细胞，分别用溶剂对照（含0.1%牛血清白蛋白（Bovine Serum Albumin，BSA）的磷酸盐缓冲液（Phosphate Buffered Saline，PBS））、β干扰素（interferon-beta，IFN-β）以及γ干扰素（interferon-gamma，IFN-γ）处理，并于刺激36小时后通过定向双端RNA测序完成转录组分析。