L1-GFP FACS transposition analysis

H9_hESCs were depleted in HERVH expression with shRNA via transient transfection. Cells were co-transfected with L1-GFP transposition reporter. GFP-positive cells can be detected only if L1 goes through the whole round of retrotransposition. GFP-positive cells were detected via FACS. The relative percentage of GFP-positive cells is significantly higher in HERVH-depleted H9_hESCs, while the mean GFP signal is not.

研究人员通过瞬时转染（transient transfection）方式利用短发夹RNA（shRNA）敲低H9人胚胎干细胞（H9_hESCs）中人类内源性逆转录病毒H（HERVH）的表达。随后将细胞与L1-GFP转座报告基因（L1-GFP transposition reporter）共转染。仅当L1完成完整一轮逆转座转座（retrotransposition）过程时，方可检测到绿色荧光蛋白（GFP）阳性细胞。通过荧光激活细胞分选术（FACS，Fluorescence-Activated Cell Sorting）对GFP阳性细胞进行检测。结果显示，HERVH表达敲低的H9_hESCs中GFP阳性细胞的相对占比显著升高，但其平均GFP信号强度未出现明显变化。