Retrovirus-mediated insertional mutagenesis: phagemid rescue of flanking DNA by selecting plasmid ori.
A method for screening recombinant lambda libraries was devised to select phage containing genomic regions containing provirus insertions of retroviruses that carry the kanamycin and G418 resistance factor neo and the origin of replication derived from pBR322 (oripBR). Such recombinants are phagemids, able to replicate as bacteriophages or as plasmids under lambda repressor control. lambda repressor was cloned into a plasmid derived from pSC101 that is compatible with pBR322-derived phagemids. A strain carrying this plasmid may be used to select phagemids derived from a single proviral insertion with 100% efficiency from complex recombinant libraries. Homologous recombination between proviral long terminal repeats was observed at a rate of 10(-4)/plaque-forming unit in recABC+ strains. Despite this frequency, intact phagemids are easily recovered as phage after temperature shift to 42 degrees C. Since oripBR itself is a selectable marker in this system, the method could be applied to recover any sequence carrying the ori sequence from pBR322.
Rodgers
J R
JR
Departments of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030.
Smith
C
C
Shawar
S M
SM
Rich
R R
RR
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D010957:Plasmids; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D012641:Selection, Genetic; D014161:Transduction, Genetic
D004251:DNA Transposable Elements; D012097:Repressor Proteins