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.
Lih
C J
CJ
Institute of Molecular Biology, Academia Sinica, Nankang Taipei, Taiwan, ROC.
Wu
S M
SM
Lin-Chao
S
S
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D016678:Genome; D019868:Kanamycin Kinase; D008322:Mammals; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics; D015183:Restriction Mapping; D012190:Retroviridae; D018243:Teratocarcinoma / Q000235:genetics; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D016662:Virus Integration
D017931:DNA Primers; D004273:DNA, Neoplasm; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase