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Sorting vector producer cells for high transgene expression increases retroviral titer. 2008

E L Beaudoin, and A J Bais, and R P Junghans
Boston University School of Medicine, Department of Surgical Research, North Campus, Roger Williams Medical Center, 825 Chalkstone Avenue, Providence, RI 02908, USA.

Vector producer cells are derived from helper cell lines expressing viral proteins that have been transduced to express a transgene-carrying retroviral genome. Vector producing cells express two relevant forms of RNA in their cytoplasm: vector RNA (vRNA) that is packaged as the actual gene transfer agent, and messenger RNA (mRNA) from which transgene is translated. Two premises underlie this study: (1) vRNA is limiting for virus production and (2) mRNA is proportional to vRNA. Together, these premises predict that transgene expression in the vector producing cells will be predictive of the viral titer from those cells. In this case, sorting the vector producing cells for high transgene expression should select for more virus production in vector producing cell supernatants. This prediction was supported, with a greater than fivefold benefit in viral titer. This demonstrates a rapid and simple method by which to obtain significantly increased viral titers from the same vector producing cell preparation.

UI MeSH Term Description Entries
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D005434 Flow Cytometry Technique using an instrument system for making, processing, and displaying one or more measurements on individual cells obtained from a cell suspension. Cells are usually stained with one or more fluorescent dyes specific to cell components of interest, e.g., DNA, and fluorescence of each cell is measured as it rapidly transverses the excitation beam (laser or mercury arc lamp). Fluorescence provides a quantitative measure of various biochemical and biophysical properties of the cell, as well as a basis for cell sorting. Other measurable optical parameters include light absorption and light scattering, the latter being applicable to the measurement of cell size, shape, density, granularity, and stain uptake. Cytofluorometry, Flow,Cytometry, Flow,Flow Microfluorimetry,Fluorescence-Activated Cell Sorting,Microfluorometry, Flow,Cell Sorting, Fluorescence-Activated,Cell Sortings, Fluorescence-Activated,Cytofluorometries, Flow,Cytometries, Flow,Flow Cytofluorometries,Flow Cytofluorometry,Flow Cytometries,Flow Microfluorometries,Flow Microfluorometry,Fluorescence Activated Cell Sorting,Fluorescence-Activated Cell Sortings,Microfluorimetry, Flow,Microfluorometries, Flow,Sorting, Fluorescence-Activated Cell,Sortings, Fluorescence-Activated Cell
D005822 Genetic Vectors DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition. Cloning Vectors,Shuttle Vectors,Vectors, Genetic,Cloning Vector,Genetic Vector,Shuttle Vector,Vector, Cloning,Vector, Genetic,Vector, Shuttle,Vectors, Cloning,Vectors, Shuttle
D012190 Retroviridae Family of RNA viruses that infects birds and mammals and encodes the enzyme reverse transcriptase. The family contains seven genera: DELTARETROVIRUS; LENTIVIRUS; RETROVIRUSES TYPE B, MAMMALIAN; ALPHARETROVIRUS; GAMMARETROVIRUS; RETROVIRUSES TYPE D; and SPUMAVIRUS. A key feature of retrovirus biology is the synthesis of a DNA copy of the genome which is integrated into cellular DNA. After integration it is sometimes not expressed but maintained in a latent state (PROVIRUSES). Leukemogenic Viruses,Leukoviruses,Oncornaviruses,Oncovirinae,Oncoviruses,Oncoviruses, Type C,RNA Tumor Viruses,Retroviruses,Type C Oncoviruses,C Oncovirus, Type,C Oncoviruses, Type,Leukemogenic Virus,Leukovirus,Oncornavirus,Oncovirus,Oncovirus, Type C,RNA Tumor Virus,Retrovirus,Tumor Virus, RNA,Tumor Viruses, RNA,Type C Oncovirus,Virus, Leukemogenic,Virus, RNA Tumor,Viruses, Leukemogenic,Viruses, RNA Tumor
D014776 Virus Cultivation Process of growing viruses in live animals, plants, or cultured cells. Viral Cultivation,Cultivation, Viral,Cultivation, Virus,Cultivations, Viral,Cultivations, Virus,Viral Cultivations,Virus Cultivations
D015870 Gene Expression The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION. Expression, Gene,Expressions, Gene,Gene Expressions

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