title: Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases.
abstract: One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integrase into a pseudo attP site, and then the R4 integrase was used to place a gene of interest into specific R4 target site. The authors demonstrate the successful and rapid retargeting of a G-protein-coupled receptor (cholecystokinin receptor A, CCKAR), an ion channel (the transient receptor potential cation channel, subfamily M, member 8, TRPM8), and a GFP-c-Jun(1-79) fusion protein into the specific loci in these cell lines and show that these retargeted cell lines exhibit functional and pharmacological responses consistent with those reported in the literature.
authors.affiliation: Life Technologies, Carlsbad, California 92008, USA.
authors.forename: Pauline T
authors.initials: PT
authors.lastname: Lieu
authors.forename: Thomas
authors.initials: T
authors.lastname: Machleidt
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Thyagarajan
authors.forename: Andrew
authors.initials: A
authors.lastname: Fontes
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Frey
authors.forename: Maya
authors.initials: M
authors.lastname: Fuerstenau-Sharp
authors.forename: David V
authors.initials: DV
authors.lastname: Thompson
authors.forename: Geetha M
authors.initials: GM
authors.lastname: Swamilingiah
authors.forename: Suchitra S
authors.initials: SS
authors.lastname: Derebail
authors.forename: David
authors.initials: D
authors.lastname: Piper
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Chesnut
mesh_terms: D000818:Animals; D001435:Bacteriophages / Q000201:enzymology*; D001681:Biological Assay; D015139:Blotting, Southern; D002460:Cell Line; D002999:Clone Cells; D055808:Drug Discovery / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D050053:TRPM Cation Channels / Q000378:metabolism
chemical_list: D016755:Proto-Oncogene Proteins c-jun; D050053:TRPM Cation Channels; D049452:Green Fluorescent Proteins; D019426:Integrases