Use of Zinc Finger Nuclease Technology to Knock Out Efflux Transporters in C2BBe1 Cells

Jennifer Pratt1, Neetu Venkatraman1, Amanda Brinker1, Yongling Xiao1, Jim Blasberg1, David C. Thompson1, Maureen Bourner1

1 Life Sciences R&D, Sigma‐Aldrich, St. Louis, Missouri
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 23.2
DOI:  10.1002/0471140856.tx2302s52
Online Posting Date:  May, 2012
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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. Curr. Protoc. Toxicol. 52:23.2.1‐23.2.22. © 2012 by John Wiley & Sons, Inc.

Keywords: zinc finger nuclease; genome editing; C2BBe1; Caco‐2; transporter; efflux

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Clone Generation: Nucleofection of Zinc Finger Nucleases
  • Support Protocol 1: Preparation of the Mammalian Single‐Strand Annealing (mSSA) Vector
  • Basic Protocol 2: Clone Generation: Cell Sorting and Analysis of ZFN Activity
  • Basic Protocol 3: Clone Identification: Bioanalyzer Analysis
  • Alternate Protocol 1: Clone Identification: Fluorescence‐Based Capillary Sequencing
  • Basic Protocol 4: Confirmation of Clone Identification: TA Cloning and Gene Sequencing
  • Basic Protocol 5: Validation of Drug Transporter Knockout Cell Line by Transwell Assay
  • Basic Protocol 6: Measurement of Cell Monolayer Integrity Using Lucifer Yellow
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Clone Generation: Nucleofection of Zinc Finger Nucleases

  Materials
  • Amaxa Cell Line Nucleofector Kit T (Lonza, cat. no. VACA‐1002), including Nucleofector Solution T, supplement, and specialized cuvettes
  • 20% growth medium (see recipe)
  • C2BBe1 cells (ATCC, cat. no. CRL‐2102), split 2 days prior to nucleofection and seeded in a T‐75 flask at a density that will reach 70‐80% confluency on the day of nucleofection
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • CompoZr Custom ZFNs (Sigma, cat. no. CSTZFN‐1KT) with appropriate forward and reverse primers
  • mSSA plasmid (see protocol 2)
  • 6‐well tissue culture plate
  • 50‐ml polypropylene conical centrifuge tube
  • 0.5‐ or 1.5‐ml microcentrifuge tubes
  • Nucleofector 2b Device (Amaxa)
  • 30° and 37°C incubators
  • Additional reagents and equipment for trypsinizing and counting cells ( appendix 3B)

Support Protocol 1: Preparation of the Mammalian Single‐Strand Annealing (mSSA) Vector

  Materials
  • mSSA vector backbone plasmid
  • HindIII and EcoRI with appropriate buffer(s)
  • Low‐melt agarose (e.g., SeaKem LE agarose)
  • 10× TAE buffer
  • Ethidium bromide
  • Gel extraction kit
  • Ligase and reaction buffer
  • Competent bacteria (e.g., MAX Efficiency DH5α Competent Cells, Invitrogen 18258‐012)
  • 100‐mm agar plates with 100 µg/ml ampicillin
  • LB broth
  • Carbenicillin
  • Plasmid miniprep kit
  • Endotoxin‐free plasmid maxiprep kit
  • Additional reagents and equipment for agarose gel electrophoresis, DNA ligation, and DNA transformation ( appendix 3A)

Basic Protocol 2: Clone Generation: Cell Sorting and Analysis of ZFN Activity

  Materials
  • Nucleofected cells (see protocol 1) at 70‐80% confluency in a 6‐well plate
  • Control cells (mSSA only and no plasmid)
  • Sorting buffer (see recipe), ice cold
  • 1 µg/ml propidium iodide
  • Liquidator 96 liquid handling robot (Rainin)
  • QuickExtract DNA Extraction Solution (Epicentre #QE09050)
  • 50‐ml conical centrifuge tubes
  • 30‐µm green Celltrics filter
  • Sterile polypropylene round‐bottom FACS tubes
  • 6‐, 24‐, and 96‐well flat‐bottom tissue culture plates
  • T‐225 tissue culture flasks
  • FACS Aria III (BD Biosciences)
  • Additional reagents and equipment for trypsinizing and counting cells ( appendix 3B)

Basic Protocol 3: Clone Identification: Bioanalyzer Analysis

  Materials
  • ZFN Cel1 Primers (provided in Sigma CompoZr Custom ZFN Kit, cat. no. CSTZFN‐1KT)
  • Molecular‐grade water
  • Genomic DNA from candidate clones (see protocol 3)
  • Wild‐type control DNA
  • JumpStart REDTaq ReadyMix PCR Reaction Mix (Sigma P0982)
  • GenElute PCR Clean‐Up Kit (Sigma NA1020)
  • 0.2‐ml thin‐walled PCR tubes or strip tubes
  • Thermocycler
  • NanoDrop microvolume UV‐Vis spectrophotometer
  • DNA 1000 Bioanalyzer Kit (Agilent Technologies, cat. no. 5067‐‐1504)
  • 2100 Bioanalyzer (Agilent Technologies)

Alternate Protocol 1: Clone Identification: Fluorescence‐Based Capillary Sequencing

  • Nested primers for target genes labeled with 6‐carboxyfluorescein (FAM) and hexachloro‐6‐carboxyfluorescein (HEX)
  • MicroAmp 96‐well reaction plate with rubber septa
  • PowerPlex 16 System Kit (Promega DC6531), including allelic ladder mix and internal lane standard 600
  • Hi‐Di formamide
  • Aluma Seal II film
  • 3730xl Genetic Analyzer (Applied Biosystems)
  • Peak Scanner software (Applied Biosystems)

Basic Protocol 4: Confirmation of Clone Identification: TA Cloning and Gene Sequencing

  Materials
  • PCR‐amplified DNA from clone(s) of interest (see protocol 4 or protocol 5)
  • TOPO TA Cloning Kit for Sequencing with One Shot Top10 Chemically Competent E. coli (Invitrogen K4575), including vector, salt solution, and SOC medium
  • Selective plates: 100‐mm LB agar plates with 100 µg/ml carbenicillin
  • 42°C water bath
  • Additional reagents and equipment for transforming cells ( appendix 3A)

Basic Protocol 5: Validation of Drug Transporter Knockout Cell Line by Transwell Assay

  Materials
  • C2BBe1 cells, knockout and wild type, each growing in T‐75 flasks and harvested at 90‐100% confluency
  • 0.25% trypsin/EDTA
  • 10% growth medium (see recipe)
  • Buffer B (see recipe)
  • Test compound working solution (see recipe)
  • 25 µM internal standard (IS) solution: 6.76 mg tolbutamide in 1000 ml acetonitrile
  • HPLC mobile phase A: 4 mM ammonium formate
  • HPLC mobile phase B: 4 mM ammonium formate in 90% (v/v) acetonitrile
  • 50‐ml Falcon tubes
  • 24‐Transwell plates (BD Biosciences, #351181)
  • HPLC system: e.g., Agilent 1100 with ChemStation software and Zorbax SB‐C8 column (2.1 × 50 mm, 3.5 µm)
  • Tandem mass spectrometer: e.g., ABI 4000 QTrap with Analyst 1.5 software
NOTE: For LC‐MS/MS, equivalent reagents and materials may be substituted.

Basic Protocol 6: Measurement of Cell Monolayer Integrity Using Lucifer Yellow

  Materials
  • Transwell assay samples (see protocol 7)
  • Buffer B (see recipe)
  • 0.1 mg/ml Lucifer yellow (CH dipotassium salt, Sigma L0144) in buffer B
  • 96‐well plate
  • Spectrometer
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Figures

Videos

Literature Cited

   Balimane, P.V., Han, Y., and Chong, S. 2006. Current industrial practices of assessing permeability and P‐glycoprotein interaction. AAPS J. 8:E1‐E13.
   Chen, W., Fuxing, T., Kazutoshi, H., and Borchardt, R. 2002. Caco‐2 cell monolayers as a model for studies of drug transport across human intestinal epithelium. In Cell Culture Models of Biological Barriers (C. Lehr, ed.) pp. 143‐163. Taylor & Francis, New York.
   Collin, J. and Lako, M. 2011. Concise review: Putting a finger on stem cell biology: Zinc finger nuclease‐driven targeted genetic editing in human pluripotent stem cells. Stem Cells 29:1021‐1022.
   Doyon, Y., McCammon, J.M., Miller, J.C., Faraji, F., Ngo, C., Katibah, G.E., Amora, R., Hocking, T.D., Zhang, L., and Rebar, E.J. 2008. Heritable targeted gene disruption in zebrafish using designed zinc‐finger nucleases. Nat. Biotechnol. 26:702‐708.
   Doyon, Y., Choi, V.M., Xia, D.F., Vo, T.D., Gregory, P.D., and Holmes, M.C. 2010. Transient cold shock enhances zinc‐finger nuclease‐mediated gene disruption. Nat. Methods 7:459‐460.
   Handel, E.M., Alwin, S., and Cathomen, T. 2009. Expanding or restricting the target site repertoire of zinc‐finger nucleases: The inter‐domain linker as a major determinant of target site selectivity. Mol. Ther. 17:104‐111.
   International Transporter Consortium. 2010. Membrane transporters in drug development. Nat. Rev. Drug Discov. 9:215‐236.
   Miller, J.C., Holmes, M.C., Wang, J., Guschin, D.Y., Lee, Y.L., Rupniewski, I., Beausejour, C.M., Waite, A.J., Wang, N.S., Kim, K.A., Gregory, P.D., Pabo, C.O., and Rebar, E.J. 2007. An improved zinc‐finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25:778‐785.
   Peterson, M.D. and Mooseker, M.S. 1992. Characterization of the enterocyte‐like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco‐2. J. Cell Sci. 102:581‐600.
   Shukla, V.K., Doyon, Y., Miller, J.C., DeKelver, R.C., Moehle, E.A., Worden, S.E., Mitchell, J.C., Arnold, N.L., Gopalan, S., Meng, X., Choi, V.M., Rock, J.M., Wu, Y.Y., Katibah, G.E., Zhifang, G., McCaskill, D., Simpson, M.A., Blakeslee, B., Greenwalt, S.A., Butler, H.J., Hinkley, S.J., Zhang, L., Rebar, E.J., Gregory, P.D., and Urnov, F.D. 2009. Precise genome modification in the crop species Zea mays using zinc‐finger nucleases. Nature 21:437‐441.
   Szczepek, M., Brondani, V., Buchel, J., Serrano, L., Segal, D.J., and Cathomen, T. 2007. Structure‐based redesign of the dimerization interface reduces the toxicity of zinc‐finger nucleases. Nat. Biotechnol. 25:786‐793.
   Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S., and Gregory, P.D. 2010. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11:636‐646.
   Wang, Z., Hop, C.E.C.A., Leung, K.H., and Pang, J. 2000. Determination of in vitro perm eability of drug cand idates through a Caco‐2 cell monolayer by liquid chromatography/tandem mass spectrometry. J. Mass Spectrom. 35:71‐76.
   Woodcock, S., Williamson, J., Hassan, I., and Mackay, M. 1991. Isolation and characterization of clones from the Caco‐2 cell line displaying increased taurocholic acid transport. J. Cell Sci. 98:323‐332.
Key Resource
   http://www.sigmaaldrich.com/life‐science/zinc‐finger‐nuclease‐technology.html
  Website for technical help with targeted genome editing using zinc finger nucleases.
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