Two shRNA constructs were used to knock down EphrinB2: shRNA 1 (5′-CGG-GTG-TTA-CAG-TAG-CCT-TAT-3′) and shRNA 2 (5′-CAG-ATT-GTG-TAC-ATA-GAG-CAA-T-3′), both obtained from Sigma-Aldrich (St. Louis, MO, USA). shRNA were cloned into the PLKO lentiviral vector6 (link) and infected by retrovirus into undifferentiated Ocy454 cells. Infected cells were selected with puromycin (5 µg/mL) and cultured at permissive temperature (33 °C) before transfer to 37 °C for differentiation. Knockdown was validated by qRT-PCR using multiplex primers, as above.
Other organizations :
St Vincents Institute of Medical Research, St Vincent's Hospital, University of Melbourne, Griffith University, Walter and Eliza Hall Institute of Medical Research, Okayama University, Australian Synchrotron, Australian Nuclear Science and Technology Organisation, Université de Bordeaux, Inserm
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