Designing Non-viral Targeted Integrating Vectors for Genome Engineering in Vertebrates

Abstract

Genome engineering for biomedicine and biotechnology requires the integration of transgenes at speci fi c sites in safe genomic environments. These speci fi c integrations are needed to avoid position effects, insertional mutagenesis and chromosome abnormalities. Several efforts during the last decade led to the development of nonviral approaches based on DNA-modifying enzymes by exploiting the cellular mechanisms of cut-and-paste transposition and homologous recombination (HR). These methods include the use of zinc fi nger nucleases, meganucleases, site-speci fi c recombinases such as F C31 integrase, Cre and Flp recombinases and transposasebased systems to achieve the integration of foreign DNA at a desired genomic position. Moreover, many teams are now investigating strategies to alter the site-speci fi city of these enzymes and precisely target safe insertion sites.This review attempts to provide a general overview on recent advances in designing nonviral site-directed integrating vectors, required for both gene therapy and animal transgenesis. We discuss the advantages of such engineered vectors, the strategies employed to improve their targeting ef fi ciency and consider their limitations in terms of safety and activity.

Generation of monoclonal antibody-producing mammalian cell lines

“…A gene of interest flanked by the same inverted terminal repeats, together with a helper plasmid expressing the transposase, mediates transposition of the gene on the donor plasmid onto several possible sites in the chromosome [146,147]. This method is of interest to mAb cell line generation as most natural transposon sites are highly active.…”