A Baculovirus-Cre/1oxP Hybrid System for AAVS1 Locus-Directed Transgene Delivery

Abstract

Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Homologous recombination is first employed in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that averts transgene silencing phenomena. Cre recombinase mediated cassette exchange is then performed using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in the differentiated progenies. Thus, this study demonstrates the feasibility of genome manipulation at the AAVS1 locus through the use of a baculovirus-Cre/loxP hybrid system. The technique developed will be useful for repeated gene targeting at a defined locus of the hESC genome.