Generation of BAC reporter cell lines for drug discovery

Betty R. Kao, Bradley McColl, Jim Vadolas

Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Otherpeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationBacterial Artificial Chromosomes
Subtitle of host publicationSecond Edition
PublisherSpringer
Chapter18
Pages323-343
Number of pages21
Volume1227
ISBN (Electronic)9781493916528
ISBN (Print)9781493916511
DOIs
Publication statusPublished - 1 Jan 2015
Externally publishedYes

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
ISSN (Print)1064-3745

Keywords

  • Bacterial artificial chromosomes
  • Cellular assay system
  • Drug discovery
  • Fluorescent reporter cell line
  • High-throughput screening
  • Stable transfection

Cite this

Kao, B. R., McColl, B., & Vadolas, J. (2015). Generation of BAC reporter cell lines for drug discovery. In Bacterial Artificial Chromosomes: Second Edition (Vol. 1227, pp. 323-343). (Methods in Molecular Biology). Springer. https://doi.org/10.1007/978-1-4939-1652-8_18