Phage integrases for the construction and manipulation of transgenic mammals.

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the phiC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos. Transgenic early embryos and a mid-gestation mouse are reported. We also demonstrate the ability of the phiC31, R4, and TP901-1 phage integrases to recombine two introduced att sites on the same chromosome in human cells, resulting in deletion of the intervening material. We compare the frequencies of mammalian chromosomal deletion catalyzed by these three integrases in different chromosomal locations. The results reviewed here introduce these bacteriophage integrases as tools for site-specific modification of the genome for the creation and manipulation of transgenic mammals.

Extrachromosomal plasmid vectors for gene therapy.

Extrachromosomal DNA is becoming widely utilized as a gene therapy vector. Plasmid DNA offers multiple advantages over viral gene therapy vectors, including large packaging capacity, stability without integration and reduced toxicity. Furthermore, plasmid DNA can be delivered to many different tissues, using a variety of delivery techniques currently being developed. This review will discuss the advantages of extrachromosomal DNA as a gene therapy vector, highlighting recent advances and successes in its use in vivo.

Phage TP901-1 site-specific integrase functions in human cells.

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.