ABSTRACT
Objective:To explore the ethical review questions of CRISPR/Cas9 gene editing technology caused in clinical research,and thus to provide a reference for matters needing attention of the ethical review involved in this technology in clinical.Methods:This paper summarized the ethical problems of CRISPR/Cas9 gene editing technology at home and abroad,analyzed the reasons and put forward some suggestions for the application of new technology in line with China's national conditions.Results:It should allow CRISPR/Cas9 gene editing technology to be applied in somatic cell gene therapy,forbidden for genital gene therapy and not considered to enhance.Since CRISPR/Cas9 lack clear subject of responsibility ethics,it brings security,conflict of rights and social equality issues.The measures that need to be taken include the strengthening of cultural communication,the formation of ethic forms of gene editing technology,the establishment of independent ethical review body at national's level,the improvement of legal norms,the formulating of technical standards and ethical principles and the major support to gene editing research field at the national level.Conclusion:In view of the potential clinical application of CRISPR/Cas9,our country should progressively restrictively develop the embryonic gene editing techniques from the prohibition.The ethics committee is responsible for the ethical review and supervision of clinical research.Members of the ethics committee and ethical staff should strengthen the study of new knowledge,strictly docking policies and regulations from accepting the clinical research projects involving CRISPR / Cas9 to ethical review,and thus to ensure the effectively review the ethical problems of genetic editing technology project.
ABSTRACT
Objective To construct Nedd4 knockout bone marrow-derived macrophages(BMDM) cell line by CRISPR/Cas9 technology and to provide an effective tool for studying the function and mechanism of Nedd4 in macrophage.Methods First,three high-grade sgRNAs targeting Nedd4 gene exons were screened using the online tool before synthesized sgRNAs were inserted into the PX330 plasmid respectively.Secondly,the recombinant plasmids were transferred into BMDM cells and monoclonal cells were obtained by limiting dilution method.The protein levels of NEDD4 in monoclonal cells were detected by Western blotting.Finally,the DNA sequence of the monoclonal cells was confirmed by sequence analysis.Results One Nedd4 knockout BMDM cell line was obtained.The sequencing result showed that the Nedd4 gene had 16bp deletion mutation in this cell line.Conclusion The Nedd4 knockout BMDM macrophage cell line constructed by CRISPR/Cas9 technology will be a useful tool for studying the function and mechanism of Nedd4 in BMDM cells.