Generation of site-specific retargeting platform cell lines for drug discovery using phiC31 and R4 integrases.
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.
Lieu
Pauline T
PT
Life Technologies, Carlsbad, California 92008, USA.
Machleidt
Thomas
T
Thyagarajan
Bhaskar
B
Fontes
Andrew
A
Frey
Elizabeth
E
Fuerstenau-Sharp
Maya
M
Thompson
David V
DV
Swamilingiah
Geetha M
GM
Derebail
Suchitra S
SS
Piper
David
D
Chesnut
Jonathan D
JD
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
D016755:Proto-Oncogene Proteins c-jun; D050053:TRPM Cation Channels; D049452:Green Fluorescent Proteins; D019426:Integrases