Selection for retroviral insertions into regulated genes

Abstract

Gene traps can be used to monitor faithfully the changes in gene expression accompanying several cellular processes. Here, we present a strategy that combines retroviral gene trap vectors, efficient selection schemes based on fluorescence-activated cell sorting or dominant positive and negative drug selection, and appropriately responsive cell lines in order to enrich for retroviral insertions into regulated genes (i.e., genes participating in cellular differentiation processes and genes induced by growth factors, drugs, or neurotransmitters, etc.). As an example, we applied this approach to the identification of insertions into genes activated by a MyoD protein, using a MyoD-responsive fibroblast line. In a single experiment designed to demonstrate the feasibility of this approach, we have been able to screen thousands of gene trap integrations and to select those that represent direct or indirect targets of MyoD. Distinct patterns of regulation were observed during myogenic determination. Sequences flanking the integrations can be rescued with several approaches, and they can be used to isolate the host genes or can serve as entry points for genome-wide sequencing projects.

A new positive/negative selectable marker, puΔtk, for use in embryonic stem cells

“…Positive/negative selectable markers such as HyTK and Tkneo have been utilized as reporter genes in genetrapping vectors. These dual-selection cassettes allow one to trap genes that can be induced or repressed in response to specific signals (Gogos et al, 1997;Harrison and Miller, 1996;Muth et al, 1998). However, retaining either HyTK or Tkneo in the ES cell clone with a trap of interest will preclude germ-line transmission of these clones because of the sterility caused by cryptic expression of HSV1 tk in the testes (Al-Shawi et al, 1988Braun et al, 1990;Dzierzak et al, 1993;Heyman et al, 1989;Iwakura et al, 1988;Neznanov et al, 1993).…”