RPS14基因RNAi慢病毒载体构建及在SKM-1细胞中的表达

Construction of RNA interference lentiviral vector of ribosomal protein S14 gene and the expression in SKM-1 cells

背景:有研究表明,造血干/祖细胞内RPS14表达减少可导致造血细胞病态造血,尤其是向红系分化受阻,提示RPS14的正常表达有助于维持机体的正常造血。目的:构建RPS14基因RNAi慢病毒载体,并观察该基因在人骨髓增生异常综合征细胞株SKM-1细胞中的表达。方法:针对已经筛选确定的RPS14基因RNAi有效靶序列,构建GC-shRPS14慢病毒载体并测序鉴定。用GC-shRPS14、pHelper1.0载体和pHelper2.0载体共转染包装细胞293T细胞,包装产生慢病毒,测定病毒滴度;并将获得的RPS14shRNA慢病毒载体GC-shRPS14转染人骨髓增生异常综合征细胞株SKM-1,用Western Blot检测靶细胞内RPS14蛋白的表达。结果与结论:经测序证实,构建出了RPS14shRNA的慢病毒载体GC-shRPS14。包装慢病毒,浓缩病毒悬液的滴度为2×109TU/mL。荧光显微镜下能直接观察到转染组细胞的GFP表达,转染效率为75%,WesternBlot检测到转染后RPS14在靶细胞中表达明显被抑制。说明成功构建RPS14基因RNAi慢病毒载体,转染人SKM-1细胞株后RPS14蛋白表达沉默。

BACKGROUND： Studies have shown that decreased expression of ribosome protein S14 （RPS14） gene in hematopoietic stem/progenitor cells can lead to the dysplasia of hematopoietic cells, in particular the blocked erythroid differentiation. Normal expression of RPS14 gene can help to maintain the normal hematopoiesis. OBJECTIVE： To construct a RNA interference lentiviral vector of RPS14 gene, and to observe its expression in the human myelodysplastic syndrome cell line SKM-1 cells. METHODS： The targeting sequence of RPS14 gene which effectively silenced in RNA inference was confirmed in our previous study. The GC-shRPS 14 lentiviral vector was established and sequenced. The 293T cells were co-transfected with lentiviral vector GC-shRPS14, pHelper 1.0 and pHelper 2.0. The titer of virus was tested according to the expression level of GFP. The resulting S14shRNA lentiviral vector GC-shRPS14 was used to infect human myelodysplastic syndrome cell line SKM-I. And the RPS14 gene expression in the target cells was detected by Western Blot. RESULTS AND CONCLUSION： DAN sequencing demonstrated that the lentiviral vector GC-shRPS14 of RPS14 shRNA was constructed. The titer of concentrated virus was 2×10^9Tu/ml. GFP expression could be seen in the transfected SKM-1 cells under fluorescence microscope, and the transfection efficiency was 75% Western Blot showed that. RPS14 expression was significantly inhibited in the target cells after transfection. The RNA interference lentiviral vector of RPS14 was constructed successfully, which is capable of delivering the target gene RPS14 into human myelodysplastic syndrome cell line SKM-1 for its silent expression.