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Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

s human tissues or cell lines by two cycles of purification with oligo dT-cellulose. Double-stranded cDNA was synthesized using Stratagenes cDNA synthesis kit, and size-selected cDNA was inserted between the EcoR I and Xho I sites of the plasmid pFB (Figure 1A). The pFB vector has an extended packaging site and uses the viral splice donor and splice acceptor configuration from the high-titer, high-expressing vector pMFG.7 To minimize packaging size constraints, and because the high infection efficiency of the system obviates the need for selection of transduced cells, there are no selectable markers in this vector. To assess the quality of both the phosphatase-treated EcoR I/Xho I-digested vector as well as the quality of the Daudi cDNA, XL10-Gold ultracompetent cells were transformed with ligated plasmid. Miniprep DNA from 100 of the resultant colonies was digested and analyzed. All of the 100 clones examined had EcoR I/Xho I-excisable inserts, with an average size of 1.7 kb and a size range of 0.5 to 5.0 kb (data not shown). Primary clones (1.1 x 106) were amplified to construct the pFB Daudi library. Figure 2 shows the size distribution for 20 representative cDNAs from this library.

Fig.2

Functional Cloning Strategy

In the schematic showing a general strategy for functional cloning using ViraPort libraries (Figure 1B), high-titer viral stocks were transiently produced by transfecting a 293 cell-based retroviral producer line 8,9 with the plasmid cDNA library, then harvesting cell supernatants after 2 days. Alternatively, 293 or 293T cells (ATCC) may be cotransfected with the plas
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