ViraPort Retroviral Gene Expression Systems - Details & Specifications

Expression cloning enables the identification and isolation of a gene based on its function or phenotype in the absence of any prior knowledge of the sequence of the nucleic acid or protein. The use of retroviral vectors for expression cloning has several advantages over traditional methods. Recent advances in viral packaging systems ensure that virtually any mitotic cell type can be transduced with efficiencies approaching 100%. The copy number of individual cDNA expression cassettes can be easily controlled by varying the multiplicity of infection (MOI). Thus, populations of infected cells may be generated in which greater than 90% of the cells are transduced with 1–5 individual cDNAs per cell, greatly reducing the time and labor of isolating the gene of interest. A wide range of gene types have been cloned from complex cDNA libraries using functional assays that allow selection or screening for a specific trait.

The high titer viral stock provided was produced from a cDNA plasmid library of high complexity inserted directionally into the replication-defective retroviral vector pFB. The target cell line of choice is transduced by simply thawing the viral supernatant, diluting in media for infection at the desired multiplicity, and adding the diluted virus directly to the cells. Two or more days following infection an appropriate selection or screen is applied to the cells, and positive transduced cells are clonally expanded. The cDNA insert is then recovered from isolated genomic DNA by PCR using the vector-specific primers provided, and subcloned for sequencing and further functional validation. If desired, the recovered cDNA may be used as probe to re-isolate clones directly from the original plasmid library from which the viral stock was derived. Plasmid libraries are available separately as E. coli glycerol stocks.

The ViraPort retrovirus was produced by transiently transfecting tissue culture cells with the pFB XR plasmid cDNA library together with two additional vectors from which the Gag-Pol and VSV-G proteins are expressed (pVPack-GP and pVPack-VSV-G, respectively). Because all of the cis and trans elements required to produce infectious virus are separated onto three plasmids, with minimal or no sequence overlap between the plasmids, there is a relatively low probability of production of replication-competent retrovirus (RCR) due to homologous recombination between the vectors. This viral production system is considerably safer than the majority of stable producer cell lines or vector-based systems for which there is a large degree of homology between the packaging vector(s) and the retroviral expression vector.

For Research Use Only. Not for use in diagnostic procedures