| Products/Services Used | Details | Operation |
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| PCR Cloning and Subcloning | pCMV-gag-pol and pMD2.G (GenBank: MS855620.1) were kindly provided by Dr. Frank J. Rauscher (The Wistar Institute, Philadelphia, USA). A retroviral transfer vector pLEGFPC1 (coding for green fluorescent protein) was used as a positive control. To obtain a high and stable recombinant protein production in CHO cells, a retroviral transfer plasmid, pLPCX (Clontech), was modified by replacing the human CMV promoter with the murine CMV promoter (mCMV) upstream of the multiple cloning site (MCS) and insertion of a woodchuck post-transcriptional regulatory element immediately downstream of the MCS. The new plasmid was designated as pRDM. A GLP-1-Fc expression cassette was synthesized by GenScript (Nanjing, China) and subcloned into pRDM to obtain pRDM-GLP-1-Fc. All constructs were confirmed by restriction enzyme digestion and DNA sequencing. | Get A Quote |
Background Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then u... More
