( mid library in addition to o...)Functional Cloning Using,,,ViraPort Retroviral cDNA Expression Libraries

mid library in addition to one or more expression vectors that provide the viral structural proteins gag, pol, and env in trans.^10,11 We used the former method for this study and achieved a titer of approximately 10⁷ cfu/ml, as determined by RT-PCR using the prostar HF RT-PCR system¹² and vector-specific primers. After an additional one or more days, cells were selected or screened for the desired phenotype, and colonies or selected populations were expanded and either enriched by one or more cycles of selection or analyzed directly. Genomic DNA was then isolated, and the cDNA inserts were retrieved by PCR using vector-specific primers that flank the insert. The PCR products were then subcloned into high-efficiency PCR cloning vectors (e.g., PCR-Script or pCMV-Script vectors) that allow sequencing of the insert and further functional analysis.

Expression Screening for Oncogenes

The ability of oncogene expression to confer deregulated growth on certain contact-inhibited cell lines, such as NIH3T3 fibroblasts, is a proven, powerful tool for identifying and isolating natural oncogenes, as well as genes involved in signal transduction pathways and cell cycle regulation.¹³ Constitutive expression or truncation of many of these latter classes of genes leads to a loss of contact inhibition and, thus, focus formation in NIH3T3 cells.

Table 2: Complexity of the Daudi pFB cDNA Expression Library

Focus #

Insert size (kb)

cDNA recovered

'"/>

Source:


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