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Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

and rescreening before the gene of interest is identified.

The use of retroviral vectors for expression cloning has several advantages over traditional methods. Recent advances in viral packaging systems ensure that virtually any cell type can be transduced with efficiencies approaching 100%.2 Additionally, the copy number of individual cDNA expression cassettes can be easily controlled by varying the MOI. Hence, populations of infected cells may be generated in which greater than 90% of the cells are transduced with one-to-five individual cDNAs per cell, greatly reducing the time and labor required to isolate the gene of interest.3 The Moloney Murine Leukemia Virus (MMLV)-based vectors have a large insert capacity (8.0 kb).4 The retroviral vectors integrate into transcriptionally active regions of the host genome, and two days following infection, a collection of stable cells are produced in which there is minimal clonal variegation of expression from cell to cell. Finally, improved transfection methods have allowed transient production of high-titer viral supernatants containing representative mixtures of cDNAs without the concern of clonal skewing, which likely occurs with the production of stable producer cell lines.5

Fig.1

We have produced ViraPort retroviral cDNA expression libraries in the high-titer vector pFB6 (Figure 1A). This vector is an MMLV-based vector in which titers of 107 to 108 colony forming units (cfu)/ml are routinely achieved using 293 cell-based transient packaging systems. We validated the use of ViraPort libraries for functional cloning by screening the human Burkitts Lymphoma cDNA library for oncogenic cDNAs.

cDNA Library Construction

Polyadenylated mRNA was isolated from variou
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