Characterization of retroviral vector derived DNA-isoforms by PCR and sequencing

For the analytical surveillance of genetic engineering operations suitable methods for characterization of used (recombinant) cell lines and used transfer vector plasmids are needed. The purpose of the described method is the identification of retroviral DNA-isoforms based on lenti-or gammaretrovirus (HIV-1 or MLV). The primers, used in an optimized touch-down PCR protocol, specifically amplify transfer-vector encoded vector genomes and proviruses integrated into stably transduced vertebrate cell lines, distinguishing them from the background of endogenous retroviral sequences. The resulting PCR products were isolated by preparative agarose electrophoresis and subsequently used for sequence identification by Sanger sequencing. In addition, the amplification and sequencing of PCR products corresponding to the HIV-1- and MLV-based vector-genomes was successful from residual transfer vector plasmids in cell culture supernatants and basic polyethylene glycol-precipitated vector preparations. The methods were validated using 19 samples (cell lines, plasmids and viral vector preparations). No false-positive PCR results were observed, while both retroviral DNA-isoforms were detected with the respective assay. The successful implementation in routine analysis is presented using two routine samples.     «

For the analytical surveillance of genetic engineering operations suitable methods for characterization of used (recombinant) cell lines and used transfer vector plasmids are needed. The purpose of the described method is the identification of retroviral DNA-isoforms based on lenti-or gammaretrovirus (HIV-1 or MLV). The primers, used in an optimized touch-down PCR protocol, specifically amplify transfer-vector encoded vector genomes and proviruses integrated into stably transduced vertebrate cel...     »