http://www.omnilytics.com/documents/What Makes Bacteriophage Safe.pdf
What Makes Bacteriophage Safe?
http://www.pnas.org/content/97/11/5995.full.pdf
A phage integrase directs efficient site-specific integration in human cells
Amy C. Groth*, Eric C. Olivares*, Bhaskar Thyagarajan, and Michele P. Calos†
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120
Edited by Allan Campbell, Stanford University, Stanford, CA,
approved March 2, 2000 (received for review December 3, 1999)
The integrase from the Streptomyces phagefC31 carries out efficient recombination between theattPsite in the phage genome and theattBsite in the host bacterial chromosome. In this paper,
we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular inte-gration ofattPintoattB. T
his assay was used to demonstrate that
in the presence of the fC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes ofattBandattPat 34 bp and 39 bp, respectively.Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attPinto an established Epstein–Barr
virus plasmid bearingattBwas documented in human cells.
This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis
for site-specific integration strategies potentially useful in a broad range of genetic engineering applications
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC135144/
J Bacteriol. 2002 July; 184(13): 3657–3663.
Phage TP901-1 Site-Specific Integrase Functions in Human Cells
Stephanie M. Stoll,
Daniel S. Ginsburg, and
Michele P. Calos*
Abstract
We demonstrate that the site-specific integrase encoded by phage TP901-1 of
Lactococcus lactis subsp.
cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in
Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in
E. coli and determined that for complete reaction in
E. coli, the minimal sizes of
attB and
attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC433029/
Proc Natl Acad Sci U S A. 1975 September; 72(9): 3531–3535.
Gene transfer to human cells: transducing phage lambda plac gene expression in GMI-gangliosidosis fibroblasts.
J Horst,
F Kluge,
K Beyreuther, and
W Gerok
Abstract
Genetic information from the bacterium Escherichia coli was transferred to human cells by means of the specialized transducing phage lambda plac carrying the bacterial z gene for the enzyme beta-galactosidase (geta-D-galactoside galactohydrolase, EC 3.2.1.23). As recipient cells, cultured skin fibroblasts from a patient with generalized gangliosidosis (GMI-gangliosidosis Type I) characterized by a severe deficiency of beta-galactosidase activity were used. The deficient human cells were incubated with the bacteriophage lambda plac or lambda plac DNA and beta-galactosidase activity was measured in order to detect gene transfer and acceptance of the prokaryotic information in the mammalian system for transcription and translation. The expression of the phage genome in the deficient fibroblasts could be demonstrated by detection of higher beta-galactosidase activity after incubation with phage lambda plac in three out of 19 experiments and in four out of 16 experiments after treatment with lambda plac DNA. Lambda plac DNA induced much higher enzyme activities than infective phage particles. Immunochemical and physicochemical assays could not distinguish the induced beta-galactosidase activity from that of the z-gene product of E. coli.