SELECTION FOR RETROVIRAL INSERTIONS INTO REGULATED GENES

Gene traps can be used to monitor faithfully the changes in gene expre ssion accompanying several cellular processes. Here, we present a stra tegy that combines retroviral gene trap vectors, efficient selection s chemes based on fluorescence-activated cell sorting or dominant positi ve and negative drug selection, and appropriately responsive cell line s in order to enrich for retroviral insertions into regulated genes (i .e., genes participating in cellular differentiation processes and gen es induced by growth factors, drugs, or neurotransmitters, etc.). As a n example, we applied this approach to the identification of insertion s into genes activated by a MyoD protein, using a MyoD-responsive fibr oblast line. In a single experiment designed to demonstrate the feasib ility of this approach, me have been able to screen thousands of gene trap integrations and to select those that represent direct or indirec t targets of MyoD. Distinct patterns of regulation were observed durin g myogenic determination. Sequences flanking the integrations can be r escued with several approaches, and they can be used to isolate the ho st genes or can serve as entry points for genome-wide sequencing proje cts.