Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity

10.1038/gt.2014.11Gene Therapy214402-412GETH

Site-specific recombinases for manipulation of the mouse genome.

Site-specific recombination systems are widespread and popular tools for all scientists interested in manipulating the mouse genome. In this chapter, we focus on the use of site-specific recombinases (SSR) to unravel the function of genes of the mouse. In the first part, we review the most commonly used SSR, Cre and Flp, as well as the newly developed systems such as Dre and PhiC31, and we present the inducible SSR systems. As experience has shown that these systems are not as straightforward as expected, particular attention is paid to facts and artefacts associated with their production and applications to study the mouse genome. In the next part of this chapter, we illustrate new applications of SSRs that allow engineering of the mouse genome with more and more precision, including the FLEX and the RMCE strategies. We conclude and suggest a workflow procedure that can be followed when using SSR to create your mouse model of interest. Together, these strategies and procedures provide the basis for a wide variety of studies that will ultimately lead to the analysis of the function of a gene at the cellular level in the mouse