title: Use of zinc finger nuclease technology to knock out efflux transporters in C2BBe1 cells.
abstract: A limitation of the traditional Caco-2 cell assay for measuring transporter-mediated efflux of a given substrate is that it is not possible to determine which specific transporter is involved. The methods in this unit describe an approach for generating specific transporter knockout cell lines that can be used to test efflux with any desired substrates. In this approach, human C2BBe1 cells (a subclone of Caco-2 cells) are nucleofected with specific zinc finger nucleases (ZFN), which can be designed to target any gene of interest and generate a double-stranded break. The cell's normal repair mechanisms can then generate targeted deletions (or integrations). A single ZFN can be used to generate a single transporter knockout, or multiple ZFNs can be used to knock out more than one transporter. This unit provides all methods needed to design the required plasmids, generate and identify transporter knockout cell lines, verify their membrane integrity, and test them with functional transport assays.
authors.affiliation: Life Sciences R&D, Sigma-Aldrich, St. Louis, Missouri, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Pratt
authors.forename: Neetu
authors.initials: N
authors.lastname: Venkatraman
authors.forename: Amanda
authors.initials: A
authors.lastname: Brinker
authors.forename: Yongling
authors.initials: Y
authors.lastname: Xiao
authors.forename: Jim
authors.initials: J
authors.lastname: Blasberg
authors.forename: David C
authors.initials: DC
authors.lastname: Thompson
authors.forename: Maureen
authors.initials: M
authors.lastname: Bourner
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D010957:Plasmids; D014162:Transfection; D016335:Zinc Fingers
chemical_list: D018528:ATP-Binding Cassette Transporters; D049452:Green Fluorescent Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific