title: Gain-of-function somatic cell lines for drug discovery applications generated by homologous recombination.
abstract: Gene targeting allows for precise genomic engineering and has been used extensively to generate both loss-of-function and gain-of-function models in mice. Similar manipulation of the genome of somatic cell lines holds high value in basic and applied research, but has been hampered by low recombination frequencies and the subsequent labor-intensive analysis of a large number of cell clones. By combining gene targeting methods with fluorescence-activated cell sorting, gain-of-function cell lines were generated and identified based on a functional readout. To demonstrate the general applicability of this approach to drug discovery, we generated targeted promoter insertion cell lines for two key drug target classes -- the G protein-coupled receptor melanocortin-receptor 4 and the nuclear receptor peroxisome proliferator-activated receptor-gamma. Molecular analysis of the engineered cell clones confirmed the predicted integration of a constitutive promoter into an endogenous allele, and the appropriate pharmacology for these targets validated the use of these gain-of-function cell lines in drug discovery applications, including high-throughput compound screening.
authors.affiliation: Vertex Pharmaceuticals--San Diego, CA, USA.
authors.forename: Karin
authors.initials: K
authors.lastname: Zeh
authors.forename: Pamela
authors.initials: P
authors.lastname: Sanders
authors.forename: Phillip
authors.initials: P
authors.lastname: Londo
authors.forename: James J
authors.initials: JJ
authors.lastname: Crute
authors.forename: Brian A
authors.initials: BA
authors.lastname: Pollok
authors.forename: Michael A
authors.initials: MA
authors.lastname: Whitney
mesh_terms: D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D018390:Gene Targeting / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D006822:Hybrid Cells / Q000187:drug effects / Q000502:physiology*; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics*; D013678:Technology, Pharmaceutical / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics; D000521:alpha-MSH / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C470072:MC4R protein, human; D044105:Receptor, Melanocortin, Type 4; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D000521:alpha-MSH; C027756:MSH, 4-Nle-7-Phe-alpha-