Identification of the coiled-coil domain as an essential element of MBD3 for preserving lineage commitment potential of ESCs

We explored the effect of knockout of Mbd3 gene on global expression profile in ESCs and compared the ability to counteract such alteration among three isoforms of MBD3, i.e. MBD3a, b and c. ESCs that are completely null for MBD3 (Mbd3abc-KO ESCs) were generated using EBRTcH3 ESCs as wild-type ESCs. Expression vectors for either MBD3a, b or c or one of deletion mutants of MBD3a in MBD and/or coiled-coil domains were stably introduced into Mbd3abc-KO ESCs and then such Mbd3abc-KO ESCs bearing one of transgenes were cultured with or without LIF for 12 days. Mbd3abc-KO ESCs bearing the empty vector was also generated as a control.