Establishment of a human embryonic stem cell line with stable β-catenin silencing by shRNA lentivirus.
- Author:
Yi SUN
1
;
Sicong ZENG
;
Guangxiu LU
;
Ge LIN
Author Information
- Publication Type:Journal Article
- MeSH: Cell Line; Embryonic Stem Cells; cytology; metabolism; Genetic Vectors; Humans; Lentivirus; genetics; Plasmids; RNA Interference; RNA, Small Interfering; genetics; Transfection; Wnt Signaling Pathway; beta Catenin; genetics; metabolism
- From: Journal of Southern Medical University 2012;32(8):1088-1092
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo establish a human embryonic stem cell line with stably β-catenin gene silencing by lentivirus-mediated shRNA interference.
METHODSPLKO.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.
RESULTSInfection 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.
CONCLUSIONLentiviral vector-delivered shRNA results in effective and stable β-catenin gene silencing in human embryonic stem cells.