Plug and play modular strategies for synthetic retrotransposons.

Recent progress in L1 biology highlights its role as a major driving force in the evolution of mammalian genome structure and function. This coincides with direct confirmation of the preponderance of long interspersed elements in mammalian genomes at the nucleotide level by large scale sequencing efforts. Two assay systems have been prominently featured in L1 studies over the past decade, which are used to assess L1 activities in cultured cells and transgenic mice respectively. However, constructing retrotransposon assay vectors and subsequent mapping of integration sites remain technically challenging aspects of the field. Synthetic biology approaches have changed the playing field with regard to the strategic design of retrotransposons. To streamline the construction and optimization of synthetic retrotransposons, we have implemented a highly efficient modular design for L1 vectors allowing "plug and play" swapping of individual modules as new knowledge is gained and optimization of constructs proceeds. Seven functional modules are divided by strategically placed unique restriction sites. These are utilized to facilitate module exchange and construction of L1 vectors for gene targeting, transgenesis and cell culture assays. A "double SfiI" strategy utilizing two non-complementary overhangs allows insert swapping to be carried out with a single, robust restriction/ligation cycle. The double-SfiI strategy is generic and can be applied to many other problems in synthetic biology or genetic engineering. To facilitate genomic mapping of L1 insertions, we have developed an optimized inverse PCR protocol using 4-base cutters and step-down cycling conditions. Using this protocol, de novo L1 insertions can be efficiently recovered after a single round of PCR. The proposed modular design also incorporates features allowing streamlined insertion mapping without repeated optimization. Furthermore, we have presented evidence that efficient L1 retrotransposition is not dependent on pCEP4 conferred autonomous replication capabilities when a shortened puromycin selection protocol is used, providing a great opportunity for further optimization of L1 cell culture assay vectors by using alternative vector backbones.