EMB-003 Approach for microdeletion screening in congenital heart defects

s Aspire: ART Paving the way for new frontiers mouse embryonic development. Materials/Methods: Whole-mount in-situ hybridization was carried out as described by Correia and Conlon. Using the cDNA of mouse p19 cells as the template, DNA was amplified with the primers for mouse borealin: forward, 5′-TGCCTTCCATCCAAGAAGAG-3′ and reverse, 5′AGGTGTTGGCAGTGAAGGAC-3′. The amplified DNA was cloned into pGEM-T Easy vector (Promega) and sequenced. Hybridization was performed with a digoxygenin-labelling and detection kit (Roche). Protein extracts were separated by SDS-PAGE and detected by western blotting using the western blot kit (KPL). Human anti-borealin antibody (gift from Dr Earnshaw and Dr Chang) and anti-rabbit IgG (KPL) were used at 1:500 and 1:10,000 dilutions, respectively. Anti-GAPDH antibody, anti-mouse IgG, anti-Oct-4 antibody (Santa) and anti-survivin antibody (Chemicon) were used according to the manufacturer’s protocol. After injection with the anti-borealin antibody or rabbit IgG (Santa), zygotes were brought back into the medium for culture. Indirect immunofluorescence was performed as described previously. After injection, the zygotes in the first mitotic division were immediately fixed in 4% formaldehyde at 4°C for 30 min, followed by incubation with the anti-β-tubulin antibody (Sigma) and fluoresceinconjugated anti-mouse IgG (Sigma). DNA was stained with 0.5 μg/ml DAPI (Chemicon) in 1× PBS. Results: Borealin was highly expressed in the undifferentiated ES cells, the mouse preimplantation embryos and the embryonic brain of 8.5 dpc–9.5 dpc mouse embryo. The mouse embryos were arrested at the 2-cell or 4-cell stage with the Borealin depleted. Conclusion: The authors have indicated for the first time that borealin plays crucial roles in the early development of mouse embryos and in the self-renewal and proliferation of undifferentiated HES cells.