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Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

raf-1 oncogene

Onco-13

2.5

vav-1 oncogene

Onco-20

2.7

vav-1 oncogene

The pFB Burkitts Lymphoma (Daudi) library was screened for the presence of oncogenes by first infecting low-passage NIH3T3 cells, then isolating and expanding foci after 28 days. Uninfected NIH3T3 cells were maintained as a negative control. Focus formation was observed in both uninfected and infected cell populations. Approximately three times as many foci were observed on the infected plates, and, of those, a number of foci had unusual morphologies not observed on the control plates. Twenty-four of the largest and most aggressively growing foci on the test plates were picked and expanded. Genomic DNA was extracted from nine of the clones, and cDNA inserts were PCR-amplified, subcloned, and sequenced. Due to the relatively high MOI used in this screen, none of the foci had less than four individual cDNA inserts, and one of the foci (Onco-11, Table 2) had as many as nine individual cDNAs. Seven of the nine foci examined harbored cDNAs encoding N-terminally truncated versions of the signaling proteins raf-1 and vav-1, which have been shown to be potent transforming proteins when truncated at their N-termini.13. Onco-1 contained a gene encoding a transcriptional repressor of a known cell cycle regulatory protein. It is not surprising, therefore, that constitutive expression of this cDNA might lead to unregulated cell growth. Onco-11 contained nine distinct cDNAs, none of which were obvious candidates for oncogenic transform
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