Construction and application of gene targeting replacement vector formouse coagulation factor IX gene

查看全文 作　　者：Dai Xuming’;Xue Hong;Yang Hual;Hu Yiping’ ;Fu Jiliang (Open Laboratory of Medical Molecular Genetics, Department of Basic Medicine Science, Second MilitaryMedical University, Shanghai, 200433)(Department of Biochemistry, hong Kong University of Scienc

出　　处：《Journal of Medical Colleges of PLA(China)》索引1998年第13卷第2期,共5页

摘　　要：Objective: To construct and apply a replacement targeting vector for mouse coagulation factor IX(mFIX) gene in embryonic stem (ES) cells. Methods: Based on the cloning and structural analysis of the genomic DNA fragment of coagulation factor IX gene from 129Sv mouse genomic DNA A phage library, PMFIXDEL plasmid was designed with positive-negative-selection (PNS) strategy, and constructed with commonmolecular cloning techniques. Structure of PMFIXDEL was identified by PCR and restriction analysis. Afterelectroporation with the linearized PMFIXDEL DNA, transfected ES cells were cultured in G418/GANC drugselection medium. The recombination efficacy of this vector was tested. Results: The main components ofPMFIXDEL were two copies of negative selection gene (HSV-tk expression cassette), a positive selectiongene (Neo expression cassette), long and short homologous fragments and plasmid backbone. The introduction of negative selection gene (HSV-tk ) into the construct resulted in 24-fold increase of selection.Conclusion: An effective replacement vector for mFIX gene targeting was constructed and applied in ES cell. 关键词：GENE targeting EMBRYONIC stem cells REPLACEMENT VECTOR HEMOPHILIA B coagulation factor IX GENE

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Construction and application of gene targeting replacement vector formouse coagulation factor IX gene

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