title: Non-integrative lentivirus drives high-frequency cre-mediated cassette exchange in human cells.
abstract: Recombinase mediated cassette exchange (RMCE) is a two-step process leading to genetic modification in a specific genomic target sequence. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette flanked by compatible recombination signals and expression of the recombinase. Major technical drawbacks are cell viability upon transfection, toxicity of the enzyme, and the ability to target efficiently cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV) vector in the second step combined with promoterless trapping of double selectable markers. Additionally, recombinase expression is self-limiting as a result of the exchangeable reaction, thus avoiding toxicity. Our approach provides proof-of-principle of a simple and novel strategy with expected wide applicability modelled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening.
authors.affiliation: Viral Vector Technical Unit, Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain.
authors.forename: Raul
authors.initials: R
authors.lastname: Torres
authors.forename: Aida
authors.initials: A
authors.lastname: García
authors.forename: Monica
authors.initials: M
authors.lastname: Payá
authors.forename: Juan C
authors.initials: JC
authors.lastname: Ramirez
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D019076:Transgenes / Q000502:physiology
chemical_list: C045073:Cre recombinase; D019426:Integrases