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Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

ation, and three of which were unknown. Like Onco-1 and Onco-11, all seven of the raf-1 and vav-1 foci harbor at least three additional cDNAs (not shown in Table 2). These additional cDNAs were distinct from each other and from focus to focus, an observation expected for a complex, representative cDNA library for which no selection applies other than that for the single focus-forming oncogene.

Receptor Cloning from ViraPort Retroviral Libraries

Fig.4

It is possible to clone cell-surface receptors by expression screening, provided that a receptor ligand or receptor-specific antibody is available. We screened for the IL-4 receptor (IL-4 R), which is known to be expressed on the surface of Daudi cells. NIH3T3 cells were infected at an MOI of approximately one, and, after 2 days, the cells were labeled with receptor-specific monoclonal antibody followed by a fluorescent anti-mouse IgG secondary antibody. The cells were then FACS sorted, and the sorted population was expanded for 10 days and sorted a second time. Approximately 3% of the expanded population from the first sort was collected in the second sort (Figure 4A) and expanded an additional 10 days. FACS analysis of the twice-sorted population indicated that over 90% of the cells reacted with the receptor-specific antibody. PCR reactions were performed using vector-specific primers, which flank the cloning site, and genomic DNA was isolated from either uninfected NIH3T3 cells or from the sorted population as template. Figure 4B shows a prominent 1.7-kb band that was observed for the sorted population (Lane 3), whereas no PCR product exists for the uninfected control (Lane 2).
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