Objective To investigate the effects of dedicator of cytokinesis 1 (Dock180) knockout on proliferation and apoptosis in rat derived H9C2 cardiomyocytes and their mechanisms.Methods A single guide RNA (sgRNA) targeting rat Dock180 gene was designed and constructed using CRISPR/Cas9 system.A plasmid contained above sgRNA was packaged into lentivirus and selected to knockout Dock180 in the cardiomyocytes.A single clone of cardiomyocyte with Dock180 knockout was established.Cardiomyocytes were divided into negative lentivirus group (Cas9, A group), Dock180 knockout group (B group), Cas9 lentivirus hypoxia group (C group), Dock180 knockout hypoxia group (D group).The expression of Dock180 mRNA was examined by RT-PCR, and relevant proteins were detected by Western blot.The cell proliferation rate of the cardiomyocytes was determined by MTT, and the apoptotic rate was measured by flow cytometry.Results Dock180 mRNA and protein were absent in B andD groups.Compared with A and C groups, p-ERK1/2 and Bcl-2 protein expression and cell proliferation rate were lower in B and D groups respectively (P<0.01), while Bax protein expression and cell apoptosis rate were higher in B and D groups respectively (P<0.05, P<0.01);Compared with A group, Dock180 mRNA and protein, p-ERK1/2 and Bcl-2 proteins and cell proliferation rate were reduced, while Bax protein and cell apoptosis rate were increased in C group(P<0.05,P<0.01).Compared with B group, p-ERK1/2 and Bcl-2 proteins and cell proliferation rate were decreased, while Bax protein and cell apoptosis rate were increased in D group(P<0.05,P<0.01).Conclusions Dock180 knockout with CRISPR/Cas9 can inhibit proliferation and promote apoptosis via p-ERK1/2, Bcl-2 and Bax in H9C2 cardiomyocytes.

Objective To develop a method of studying fiber projecting in the spinal cord duiring chicken embryo development.Methods At embryonic incubation 3 day (E3), pCAGGS-green fluorescent protein (GFP) plasmid was injected into the spinal cord using in vivo electroporation.Three days after transfection (E6), GFP-positive embryos were collected under a stereo fluorescence microscope .Subsequently , the spinal cord was separated from the embryos and cut from the roof plate as an open book .After fixed with 4%paraformaldehyde ( PFA) for one hour , the opened spinal cords were used for immunohistochemistry with N-cadherin antibody and with DAPI for nuclei .Finally, the nerve fiber projecting was photographed and analyzed under a fluorescence microscope . Results Based on the opened spinal cord and immunostaining in the cryosection , we observed that the nerve fibers projected across the midline of the floor plate and reached to the sulcus terminalis along the white matter of the contra side .The immunoreaction against N-cadherin indicated that overexpression of GFP has no significant effect on chicken embryonic development .Conclusion A new method to study fiber projecting in the developing chicken spinal cord is established successfully in this study .