Rapid identification and isolation of transcriptionally active regions from mouse genomes

We report here the design, construction and testing of a self-inactivating (Sin) retrovirus promoter-trap vector suitable for identifying and isolating transcriptionally active regions from the mouse genome. When this vector, which contains the bacterial aph gene as its reporter, is integrated into a site downstream from an active host cell promoter, it expresses aph, whose product, aminoglycoside phosphotransferase, produces resistance to the antibiotic G418 in mammalian cells. The construct also contains a native aph promoter which functions in bacteria, but not in mouse cells, to express kanamycin (Km) resistance, plus an adjacent pBR322-derived replication origin. Thus, mammalian DNA segments containing actively transcribed regions flanking aph can be quickly isolated by restriction endonuclease treatment of total DNA from provirus-containing mouse cells, followed by self-ligation, transformation and Km selection of plasmids carried by bacteria transformed with this DNA. We tested this Sin retrovirus promoter-trap system by isolating eight DNA segments upstream to the provirus integration sites in the genome of virus-infected mouse F9 cells. We found that the Sin retrovirus vector produces a high yield of infectious virus particles carrying aph, and that the isolated genomic DNA fragments of F9 cells are transcriptionally active.