Figure 1

A dominant strategy for the identification of signaling molecules using a tetracycline-regulated retrovector. A replication-defective retrovector was generated in which the normal transcriptional promoters of the long terminal repeats were crippled ('dead' LTR), and a promoter (Tet-Reg) repressible by the tetracycline trans-activator protein (tTA) in the presence of a tetracycline antibiotic, such as doxycycline, was inserted upstream from cloning sites, an internal ribosomal entry sequence (IRES), and GFP cDNA. After cloning a library of mutated cDNAs into the vector, the DNA was packaged into virions, and a large population of modified Jurkat T cells (carrying the tTA) was transduced with the virions; GFP expression was used to select clones expressing a unique cDNA. Cells stimulated through their antigen receptor (TCR) were scored for retention of receptor expression and loss of induction of a molecule downstream from TCR signaling, CD69. Individual clones could be rescored for reversibility of the phenotype by comparison of results in the presence (+tet) or absence (-tet) of doxycycline. Individual, sorted clones could then be used for isolation of the cDNA cloned into the retrovector. Adapted from [2].