Establishment of a human embryonic stem cell line with stable β-catenin silencing by shRNA lentivirus / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To establish a human embryonic stem cell line with stably β-catenin gene silencing by lentivirus-mediated shRNA interference.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Lentiviral vector-delivered shRNA results in effective and stable β-catenin gene silencing in human embryonic stem cells.</p>