Gene targeting provides a powerful tool for dissecting gene function. However, repeated targeting of a single locus remains a practice mostly limited to unicellular organisms that afford simple targeting methodologies. We developed an efficient method to repeatedly target a single locus in Drosophila. In this method, which we term "site-specific integrase mediated repeated targeting" (SIRT), an...

The domestic pig is an important "dual purpose" animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in gener...

N,N'-diacetylchitobiase (chitobiase) from the marine organism Vibrio harveyi is a highly stable reporter enzyme for gene fusions. This enzyme hydrolyzes the disaccharide chitobiose to N-acetyl glucosamine. The advantages of the reporter gene encoding chitobiase (chb) are: (i) that chitobiase and N-acetyl-beta-D-glucosaminidase activities are missing in E. coli strains, (ii) chitobiase can be mo...

To establish a lysogenic lifestyle, the temperate bacteriophage φC31 integrates its genome into the chromosome of its Streptomyces host, by site-specific recombination between attP (the attachment site in the phage DNA) and attB (the chromosomal attachment site). This reaction is promoted by a phage-encoded serine recombinase Int. To return to the lytic lifestyle, the prophage excises its DNA b...

Serine integrases are bacteriophage enzymes that carry out site-specific integration and excision of their viral genomes. The integration reaction is highly directional; recombination between the phage attachment site attP and the host attachment site attB to form the hybrid sites attL and attR is essentially irreversible. In a recent model, extended coiled-coil (CC) domains in the integrase su...

To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as t...

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order a...