Sun Yi, Zeng Sicong, Lu Guangxiu, Lin Ge
Institute of Reproductive and Stem Cell Engineering, Central South University, Changsha, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2012 Aug;32(8):1088-92.
To establish a human embryonic stem cell line with stably β-catenin gene silencing by lentivirus-mediated shRNA interference.
PLKO.1-puro-β-catenin vector, a lentivirus plasmid expressing β-catenin shRNA, was packaged into 293FT cells. Human embryonic stem cells were infected with the lentivirus and the cell clones stably expressing β-catenin shRNA were selected by puromycin, with the uninfected cells and cells infected with the empty vector as the control. Real-time RT-PCR was used to evaluate the efficiency of β-catenin knocked down; β-catenin and OCT4 protein expression in the infected cells was examined using immunofluorescence assay.
Infection with β-catenin-specific shRNA lentivirus resulted in stable interference of β-catenin expression in human embryonic stem cells, which showed a β-catenin mRNA expression of only 1% of that in the uninfected cells. Infection with the empty vector produced no obvious effect on β-catenin expression compared to the uninfected cells. In the cells infected with β-catenin shRNA lentivirus, β-catenin protein expression was almost undetectable in immunofluorescence assay, while OCT4 was still expressed after the interference.
Lentiviral vector-delivered shRNA results in effective and stable β-catenin gene silencing in human embryonic stem cells.
通过慢病毒介导的短发夹RNA（shRNA）干扰建立β-连环蛋白基因稳定沉默的人胚胎干细胞系。
将表达β-连环蛋白shRNA的慢病毒质粒PLKO.1-puro-β-连环蛋白载体包装到293FT细胞中。用该慢病毒感染人胚胎干细胞，并用嘌呤霉素筛选稳定表达β-连环蛋白shRNA的细胞克隆，以未感染细胞和感染空载体的细胞作为对照。采用实时逆转录聚合酶链反应（RT-PCR）评估β-连环蛋白敲低的效率；用免疫荧光法检测感染细胞中β-连环蛋白和八聚体结合转录因子4（OCT4）蛋白的表达。
感染β-连环蛋白特异性shRNA慢病毒导致人胚胎干细胞中β-连环蛋白表达受到稳定干扰，其β-连环蛋白信使核糖核酸（mRNA）表达仅为未感染细胞的1%。与未感染细胞相比，感染空载体对β-连环蛋白表达无明显影响。在感染β-连环蛋白shRNA慢病毒的细胞中，免疫荧光法几乎检测不到β-连环蛋白蛋白表达，而干扰后OCT4仍有表达。
慢病毒载体介导的shRNA可导致人胚胎干细胞中β-连环蛋白基因有效且稳定的沉默。
