title: Generation of BAC reporter cell lines for drug discovery.
abstract: Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an example, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders.
authors.affiliation: Cell and Gene Therapy Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road, Parkville, 3052, Melbourne, VIC, Australia, betty.kao@mcri.edu.au.
authors.forename: Betty R
authors.initials: BR
authors.lastname: Kao
authors.forename: Bradley
authors.initials: B
authors.lastname: McColl
authors.forename: Jim
authors.initials: J
authors.lastname: Vadolas
mesh_terms: D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics* / Q000378:metabolism; D055808:Drug Discovery; D018274:Electroporation; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D020014:K562 Cells; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014162:Transfection; D055544:beta-Globins / Q000235:genetics* / Q000378:metabolism; D055545:gamma-Globins / Q000235:genetics* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D055544:beta-Globins; C485184:enhanced green fluorescent protein; D055545:gamma-Globins; D049452:Green Fluorescent Proteins