Current Protocols in Toxicology Volume 51 || Use of Zinc Finger Nuclease Technology to Knock Out Efflux Transporters in C2BBe1 Cells

UNIT 23.2Use of Zinc Finger Nuclease Technologyto Knock Out Efflux Transporters inC2BBe1 Cells

Jennifer Pratt,1 Neetu Venkatraman,1 Amanda Brinker,1 Yongling Xiao,1

Jim Blasberg,1 David C. Thompson,1 and Maureen Bourner1

1Life Sciences R&D, Sigma-Aldrich, St. Louis, Missouri

ABSTRACT

A limitation of the traditional Caco-2 cell assay for measuring transporter-mediated effluxof a given substrate is that it is not possible to determine which specific transporter isinvolved. The methods in this unit describe an approach for generating specific transporterknockout cell lines that can be used to test efflux with any desired substrates. In thisapproach, human C2BBe1 cells (a subclone of Caco-2 cells) are nucleofected withspecific zinc finger nucleases (ZFN), which can be designed to target any gene of interestand generate a double-stranded break. The cell’s normal repair mechanisms can thengenerate targeted deletions (or integrations). A single ZFN can be used to generate asingle transporter knockout, or multiple ZFNs can be used to knock out more thanone 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.C© 2012 by John Wiley & Sons, Inc.

Keywords: zinc finger nuclease � genome editing � C2BBe1 � Caco-2 � transporter �

efflux

INTRODUCTION

The development and functional testing of ATP-binding cassette (ABC) transporterknockout cell lines in intestinal enterocytes enables researchers to clearly establish thespecific transporter involved in efflux of a particular chemical entity. The standard assayused to determine whether a particular compound is a candidate for efflux is the differ-entiated Caco-2 cell assay (Chen et al., 2002). In that assay, a potential substrate is addedto Caco-2 cells and a Transwell assay is performed. The limitation of that assay is thatit does not reveal which specific transporter or transporters are responsible for the effluxof that substrate. For this purpose, we developed an approach to engineer and test celllines with one or more ABC transporters genetically and functionally knocked out toenable researchers to identify the individual transporter (or combination of transporters)involved in the transport of specific substrates.

The approach is based on the use of zinc finger nucleases (ZFNs), which are a classof engineered DNA-binding proteins that facilitate targeted editing of the genome bycreating double-stranded DNA breaks at user-specified locations. Double-strand breaksare important for site-specific mutagenesis in that they stimulate the cell’s natural DNA-repair processes, namely homologous recombination and non-homologous end joining(NHEJ). These processes can be harnessed to generate precisely targeted gene edits,resulting in cell lines with targeted gene deletions (knock-outs), integrations (knock-ins),or modifications, as illustrated in Figure 23.2.1.

ZFNs consist of two functional domains: a DNA-binding domain comprised of a chainof zinc finger proteins, and a DNA-cleaving domain composed of the nuclease domain

Current Protocols in Toxicology 23.2.1-23.2.22, May 2012Published online May 2012 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/0471140856.tx2302s52Copyright C© 2012 John Wiley & Sons, Inc.

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Supplement 52

Targeted GenomeEditing Using Zinc

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23.2.2

Supplement 52 Current Protocols in Toxicology

DNA-CleavingDomain

b)

DNA-BindingDomain

ZFN pair deliveredinto cell bytransfection orelectroporation

a)

ZFN pair recognizesand heterodimerizesaround target site ZFN pair makes double-

strand break anddissociates from DNA

NO REPAIR TEMPLATEco-transfected withZFN pair: 1-20% of cellsare mis-repaired resultingin a Gene Deletion.Cellular Process Used: Non-homologous end joining

REPAIR TEMPLATEco-transfected withZFN pair: 1-20% of cellscontain Gene Integrationin target site.Cellular Process Used:Homologous recombination

Nucleus

Figure 23.2.1 Illustration of gene editing procedure using zinc finger nucleases (ZFNs). Top: ZFNs arecomposed of a DNA-cleaving domain from Fok1 and a sequence-specific DNA-binding domain for targetingthe gene of interest. Bottom: DNA cleavage results in a targeted double-stranded break that can be acted uponby the cell’s natural DNA repair mechanisms. Non-homologous end joining can result in a deletion (as appliedhere for making gene knockouts), whereas homologous recombination can be used to integrate sequences.

of Fok1 (Fig. 23.2.1). The zinc finger DNA-binding proteins recognize a 3-bp target.By combining four to six zinc finger proteins, each ZFN can target and specificallybind a 12- to 18-bp sequence from the gene of interest, in this case a specific effluxtransporter sequence (e.g., MDR1 or BCRP). Importantly, the endonuclease domain ofFok1 has been reengineered to function as an obligate heterodimer in order to cleaveDNA (Miller et al., 2007). This means a pair of ZFNs is required to bind and cutthe genomic DNA at the targeted site, and this property is used to ensure specificity.The targeted sequences for each ZFN must be separated by 5-7 bp to allow formationof the catalytically active Fok1 dimer. The 24- to 36-bp DNA binding specificity andadditional positional constraints drive a very high degree of precision in genome editing.Modification of a cell line is achieved using standard procedures such as transfection,single-cell cloning, and genotyping.

This unit describes methods used to develop and test knockout cell lines for specific ABCefflux transporters. Methods for clone generation include: nucleofection of zinc fingernuclease (ZFN; Basic Protocol 1), mSSA vector construction (Support Protocol), andcell sorting and analysis of ZFN activity (Basic Protocol 2). C2BBe1 cells, a subcloneof Caco-2 cells with high brush border protein expression (Peterson and Mooseker,1992), are used to develop the knockout cell lines. The C2BBe1 cells are purchasedfrom ATCC and licensed for commercial development from NaviCyte. Subsequently,

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Current Protocols in Toxicology Supplement 52

Section I: Clone Generation

Section II: Clone Identification

Section III: Functional Analysis

Nucleofect C2BBe1 cellsusing ZFN RNA & mSSA.Culture until recovered.

Pool sort cells usingGFP�PI�.

Culture until recovered.

Re-nucleofect usingZFN RNA.

Culture until recovered.

Single cell sort usingPI�. Culture to expand.

Isolate genomic DNA.Run on capillary sequencer

or bioanalyzer.Loss of wild-type?

Topo clone & sequence.Out of frame knock out?

Expand & plateon Transwell filters.

Pass

Pass

Partial

Fail

Fail

Run in Transwell assayusing transporter

specific substrates

Run in Lucifer yellow assayfor tight junction analysis

Drop

Figure 23.2.2 Flow chart for cell line development using targeted zinc finger nucleases.


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clones are identified by bioanalysis (Basic Protocol 3) and fluorescence-based capillarysequencing (Alternate Protocol), and then subcloned by TA cloning and confirmed bysequencing (Basic Protocol 4). Finally, methods are given for functional evaluation ofthe transporter knockouts by bidirectional Transwell assays (Basic Protocol 5). In thisprotocol, transport is allowed to proceed in the presence of a test compound (transportersubstrate), transport is measured by LC-MS/MS, and the permeability coefficient (Papp

value) and efflux ratio are determined (Basic Protocol 5). Finally, a method is presentedto confirm that cell monolayer integrity has been maintained by measuring the passivepassage of Lucifer yellow (Basic Protocol 6). A flow chart for the overall process ofdeveloping and testing transporter knockout cell lines using targeted ZFNs is shown inFigure 23.2.2.

BASICPROTOCOL 1

CLONE GENERATION: NUCLEOFECTION OF ZINC FINGER NUCLEASES

This protocol is used for all nucleofections performed. In the first round of nucleofection,both the targeted ZFN plasmid and an mSSA plasmid (see Support Protocol) are nucle-ofected, resulting in cells that will be GFP positive if the ZFN plasmid is successfullyinserted. With multiple alleles, several nucleofections may be needed to achieve a fullknockout. For subsequent rounds of nucleofection, eliminate the mSSA and nucleofectonly the ZFN mRNA.

Materials

Amaxa Cell Line Nucleofector Kit T (Lonza, cat. no. VACA-1002), includingNucleofector 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 dayof nucleofection

Hanks’ balanced salt solution (HBSS; APPENDIX 2A)CompoZr Custom ZFNs (Sigma, cat. no. CSTZFN-1KT) with appropriate forward

and reverse primersmSSA plasmid (see Support Protocol)

6-well tissue culture plate50-ml polypropylene conical centrifuge tube0.5- or 1.5-ml microcentrifuge tubesNucleofector 2b Device (Amaxa)30◦ and 37◦C incubators

Additional reagents and equipment for trypsinizing and counting cells (APPENDIX 3B)

Prepare cells and reagents1. From the Nucleofector Kit T, add the entire contents of the supplement solution to

the Nucleofector Solution T. Allow to warm to room temperature.

Each nucleofection will require 100 μl supplemented nucleofection solution. Once sup-plemented, the solution can be stored for up to 3 months at 4◦C.

2. For each reaction, add 2 ml of 20% growth medium to one well of a 6-well tissueculture plate. Place in a 37◦C incubator to warm.

Six reactions is the most one should attempt at one time to avoid excessive exposure ofthe cells to nucleofection solution.

3. Harvest cells by trypsinization and determine the number of viable cells using trypanblue and a hemacytometer (APPENDIX 3B).

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Each nucleofection will require 5 × 105 cells. Cells should be >90% viable at the time ofnucleofection. If the cells are not >90% viable, split the cells and check viability at thenext passage.

4. Transfer cells to a 50-ml conical centrifuge tube and centrifuge 5 min at 91 × g,room temperature.

It is critical that cells be centrifuged and handled carefully prior to nucleofection tominimize cell death.

5. Wash cells twice with 10 ml HBSS, centrifuging as in step 4.

It is important to wash cells twice when nucleofecting with mRNA, as RNases in themedium will degrade the ZFN mRNA. If nucleofecting DNA, one wash is sufficient.

6. While centrifuging cells, prepare mRNA. For each nucleofection, combine the fol-lowing in a microcentrifuge tube in a volume no greater than 15 μl:

2 μg forward mRNA for each ZFN2 μg reverse mRNA for each ZFN4 μg mSSA plasmid.

It is important to minimize the volume of mRNA solution to avoid diluting the nucleofectorsolution.

To avoid degradation of mRNA, do not mix with cells until ready to nucleofect.

Perform nucleofection7. After the final wash, aspirate the HBSS and resuspend cells at 5 × 105 cells per

100 μl nucleofection solution by gently pipetting up and down.

It is critical to minimize up-and-down pipetting, as the cell membranes become fragile innucleofection solution. It is also critical to perform the subsequent steps quickly so thatthat cells are not exposed to nucleofection solution for long periods of time. Exposure for>20 min will result in cell death.

8. Transfer 100 μl cells to the prepared mRNA in one microcentrifuge tube and thentransfer mixture to a cuvette.

9. Quickly insert the cuvette into the slot of the Amaxa Nucleofector and press “Run”.

10. Remove the cuvette and use a Pasteur pipet to transfer ∼1 ml warm medium fromone well of the 6-well plate to the cuvette. Pipet up and down gently two times, thentransfer all of the solution back to the well of the 6-well plate.

11. Repeat until all nucleofections are complete.

12. Place cells in a 30◦C incubator for 2 days to increase the efficiency of nucleofection.

Cells may look round and not fully attached to the plate at this time.

13. Move the plate to a 37◦C incubator for 2 days and then replace medium with fresh20% growth medium when the cells have attached.

14. Allow cells to grow to 70% to 80% confluency before pool sorting after the firstnucleofection or single-cell sorting after additional subsequent nucleofections (seeBasic Protocol 2).

It may take 1-2 weeks for cells to reach this level of confluency.

For nucleofection efficiency, refer to the Lonza website for each specific cell line. Efficiencycan also be tested in the cell type of interest by adding the pGFP control (provided withthe nucleofection kit) in a control well.


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SUPPORTPROTOCOL

PREPARATION OF THE MAMMALIAN SINGLE-STRAND ANNEALING(mSSA) VECTOR

The following protocol for the construction of the mSSA reporter construct is imple-mented for all ZFN recognition sequences. The mammalian single-strand annealing(mSSA) assay was established to evaluate ZFN activity in cells using transient co-transfection with the ZFN expression vectors in C2BBe1 cells. It is similar to techniquesused in yeast SSA technology (Doyon et al., 2008; Shukla et al., 2009), but uses a re-porter construct that is compatible with the current mammalian expression system. ThemSSA plasmid backbone design originated at Sangamo BioSciences and was furtheroptimized at Sigma-Aldrich. The mSSA plasmid contains sequences recognized by zincfinger proteins specific for the genes of interest. The recognition sites are flanked bysequences expressing two portions (“GF” and “FP”) of the complete GFP protein. If theZFN plasmid is successfully transformed into the cells, it cuts the target recognition site,generating a double-stranded break. When the cell repairs the plasmid through the SSADNA repair pathway, it generates a functional GFP sequence. In this way, successfullyZFN-transformed cells (see Basic Protocol 1) can be easily selected by sorting based onGFP expression (see Basic Protocol 2).

Materials

mSSA vector backbone plasmidHindIII and EcoRI with appropriate buffer(s)Low-melt agarose (e.g., SeaKem LE agarose)10× TAE bufferEthidium bromideGel extraction kitLigase and reaction bufferCompetent bacteria (e.g., MAX Efficiency DH5α Competent Cells, Invitrogen

18258-012)100-mm agar plates with 100 μg/ml ampicillinLB brothCarbenicillinPlasmid miniprep kitEndotoxin-free plasmid maxiprep kit

Additional reagents and equipment for agarose gel electrophoresis, DNA ligation,and DNA transformation (APPENDIX 3A)

1. Synthetically construct the target oligo for cloning into the mSSA vector backboneas shown:

5′—EcoRI—NNT1NN—NNT2NN—NNT3NN—NNT4NN—HindIII—3′

a. Identify the nucleotide sequences encoding ZFN recognition and cut sites for eachtarget (T). Include 2 bp of the endogenous gene sequence flanking each side ofthe recognition site (NN).

The ZFN binding and cut site sequence, in addition to flanking endogenous sequencefor the gene target, are included with the ZFN kit product information.

b. If multiple genes are being targeted, line the sequences up one after the other, asshown for T1-T5.

c. Add appropriate restriction sites to the ends of the construct to enable cloning intothe mSSA vector.

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2. Digest the plasmid containing the synthesized oligo and the mSSA vector backboneplasmid by simultaneous digestion with EcoRI and HindIII according to the enzymemanufacturer’s recommended protocol.

3. Separate digested DNA fragments on a 1% low-melt agarose TAE gel containingethidium bromide.

4. Cut out the gel fragments containing the ZFN recognition oligo insert and the mSSAplasmid vector backbone, and purify the DNA (e.g., using a gel extraction kit).

5. Ligate the isolated insert and vector backbone according to the manufacturer’s recom-mended protocol. For a background control, include a ligation reaction that containsmSSA vector backbone DNA alone.

6. Transform ligated plasmid DNA into competent bacteria according to the manufac-turer’s recommended protocol.

7. Incubate transformed bacterial cultures on ampicillin agar plates overnight at 37oC,with controls that include bacterial growth medium alone, transformed bacteriacontaining mSSA vector backbone DNA alone (vector only control), and transformedbacteria containing mSSA vector backbone DNA with target oligo insert DNA (vectorplus insert).

8. Observe agar plates for bacterial colonies. If no colonies exist on the vector onlycontrol plate, pick 3-5 colonies from the vector plus insert plate for expansion. Ifcolonies grow on the vector only control plate, verify that at least 3× more coloniesexist on the vector plus insert plate. Pick at least 10-20 clones from the vector plusinsert plate for expansion.

If too many clones exist on control plates, cloning conditions should be optimized.

9. Grow picked colonies in 5 ml LB broth containing 100 μg/ml carbenicillin.

10. Isolate plasmid DNA from cultures using a standard miniprep method. Validateclones that contain an insert by restriction enzyme analysis.

11. Grow clones that contain the appropriate insert in 150 ml LB broth containing100 μg/ml carbenicillin. Isolate plasmid DNA using endotoxin-free maxipreptechnology.

BASICPROTOCOL 2

CLONE GENERATION: CELL SORTING AND ANALYSIS OF ZFNACTIVITY

This protocol is used for pooled cell sorting to analyze ZFN activity by GFP analysisusing the FACS Aria III. Isolation of GFP-positive cells results in an enrichment ofgenetically modified cells. The protocol also describes single-cell sorting after subsequentnucleofections to identify and establish the desired clones. The workflow for this protocolassumes successful completion of the final step in Basic Protocol 1.

Materials

Nucleofected cells (see Basic Protocol 1) at 70-80% confluency in a 6-well plateControl cells (mSSA only and no plasmid)Sorting buffer (see recipe), ice cold1 μg/ml propidium iodideLiquidator 96 liquid handling robot (Rainin)QuickExtract DNA Extraction Solution (Epicentre #QE09050)

50-ml conical centrifuge tubes30-μm green Celltrics filter


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Sterile polypropylene round-bottom FACS tubes6-, 24-, and 96-well flat-bottom tissue culture platesT-225 tissue culture flasksFACS Aria III (BD Biosciences)

Additional reagents and equipment for trypsinizing and counting cells (APPENDIX 3B)

Pool and stain cells1. Trypsinize (APPENDIX 3B) all sample wells in the 6-well plate and pipet cells up and

down six times to break up clumps.

2. Transfer cells from all wells to a single 50-ml conical tube.

3. Mix 20 μl cell suspension with 20 μl of 0.4% trypan blue and count cells using ahemacytometer (APPENDIX 3B).

4. Add 20% growth medium to the 50-ml tube to bring the cells to a rounded volume(e.g., 20-30 ml).

5. In separate 50-ml tubes, set up control samples using cells nucleofected with con-structed mSSA plasmid only (background) and cells without any plasmid (negativecontrol).

6. Centrifuge cells at 91 × g for 5 min, room temperature. Remove the supernatantcompletely with an aspirator tip.

7. Bring cells to a concentration of 1 × 106 cells/ml in cold (4◦C) sorting buffer. Mixcells well to break up the pellet and remove clumps.

The sorting buffer should be thoroughly cold and taken straight from the refrigerator.

8. Filter cells through a sterile 30-μm green Celltrics filter into a sterile polypropyleneround-bottom FACS tube.

9. Add 1 μl propidium iodide to the cells (final 1 μg/ml) and incubate 5-10 sec.

10. Add 1 ml of 20% growth medium to a well of a 24-well flat-bottom tissue cultureplate.

The cells are treated the same up to this point for either sorting method. At this point,proceed to pooled cell sorting or single-cell sorting.

Perform pooled cell sorting11. Using the FACS Aria III in “Yield” precision mode based on GFP+ and PI− sort

gates, pool sort the cells into one well of a 24-well plate containing 20% growthmedium (Fig. 23.2.3).

The yield should be between 3,000 and 10,000 cells from 3X10e6 cells, depending on thepercent of mSSA GFP+ and PI− cells. For example data, see Figure 23.2.3.

Perform single-cell sorting and establish clones12. Using the FACS Aria III in “Single Cell” precision mode based on a PI− sort gate,

sort cells into thirty to forty 96-well plates (Fig. 23.2.4).

13. Briefly centrifuge cells at 800 rpm for 5 min and then incubate at 37◦C.

Colonies will start to appear on the plates after 12-14 days.

14. Select all clones and trypsinize cells using a Liquidator 96 liquid handling robot.

15. Transfer detached cells to a new 96-well plate. Then prepare a daughter plate bytransferring half the volume of each clone to another 96-well plate.

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A

101101

eGFP-A

GFP�0.017%

PI�8.21%

Negative: No mSSA

PI-

A

102

103

104

105

102 103 104 105

B

101101

eGFP-A

GFP�1.62%

PI�20.4%

Background: mSSA without ZFNP

I-A

102

103

104

105

102 103 104 105

C

101101

eGFP-A

GFP�13.7%

PI�21.4%

Positive: mSSA with ZFN

PI-

A

102

103

104

105

102 103 104 105

Figure 23.2.3 Pooled sorting of nucleofected C2BBe1 cells using the FACS Aria III in “Yield” precision modebased on GFP+ and PI− sort gates. Results demonstrate a 12% increase in GFP fluorescence in the positivesample (nucleofected in the presence of both mSSA and ZFN plasmids).

0

0

FSC-A

PI�6.45%

PI-

A

102

103

104

105

50K 100K 150K 200K 250K

Figure 23.2.4 Single-cell sorting of nucleofected C2BBe1 cells using the FACS Aria III in “SingleCell” precision mode based on a PI− sort gate.

16. Centrifuge the daughter plate at maximum speed to pellet the cells. Remove thesupernatant and add 25 μl/well QuickExtract to prepare genomic DNA per manu-facturer’s instructions.

17. Analyze the DNA for mutations (see Basic Protocol 3).

18. Add 100 μl of 20% growth medium to the 96-well plate with cells and allow thecells to grow at 37oC while the DNA is being analyzed.


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19. Once the desired mutant clones are identified, trypsinize the corresponding cells inthe first plate (step 15), expand them to a 24-well plate, and replace 20% growthmedium with 10% complete medium.

20. Every 7-20 days, expand cells further, first to a 6-well plate, then to a T-75 flask, andeventually to a T-225 flask. Keep at least three to four mutants as backups.

21. Freeze a master cell bank for long-term storage.

BASICPROTOCOL 3

CLONE IDENTIFICATION: BIOANALYZER ANALYSIS

This protocol describes the initial screen for mutations in sorted clones. Genomic DNAis extracted from clones, scaled up by PCR amplification, and placed on bioanalyzerchips. Clone DNA is compared to wild-type DNA. If a mutation is present, at least oneband will be larger or smaller than the corresponding wild-type band.

This method uses double-stranded DNA for analysis, which can cause multiple bands toshow for a single mutation. Although the method can show that a mutation is present, itcannot predict the exact number and size of mutations. This information can be obtainedby fluorescence-based capillary sequencing as described in the Alternate Protocol.

Materials

ZFN Cel1 Primers (provided in Sigma CompoZr Custom ZFN Kit, cat. no.CSTZFN-1KT)

Molecular-grade waterGenomic DNA from candidate clones (see Basic Protocol 2)Wild-type control DNAJumpStart REDTaq ReadyMix PCR Reaction Mix (Sigma P0982)GenElute PCR Clean-Up Kit (Sigma NA1020)

0.2-ml thin-walled PCR tubes or strip tubesThermocyclerNanoDrop microvolume UV-Vis spectrophotometerDNA 1000 Bioanalyzer Kit (Agilent Technologies, cat. no. 5067–1504)2100 Bioanalyzer (Agilent Technologies)

Amplify genomic DNA using Cel1 primers1. Dilute forward and reverse ZFN Cel1 primers with molecular-grade water to 20 μM

upon receipt to make working stocks. Dilute genomic DNA from candidate clones1:10 using water. Include a known wild-type sample for analysis control.

Once diluted, all material can be stored for up to 3 months at –20◦C. Diluted genomicDNA should be treated in a manner that reduces freeze-thaw cycles.

2. Prepare PCR reactions in 0.2-ml thin-walled PCR tubes or strip tubes as follows:

20 μl JumpStart reaction mix2 μl each diluted primer (forward and reverse)16 μl diluted genomic DNA.

3. Run PCR with the following thermocycler conditions:

5 min at 94◦C35 cycles:

30 sec at 94◦C (denaturation)30 sec at 55◦C (annealing)45 sec at 68◦C (extension)

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3 min at 68◦C (extension)Hold at 4◦C.

The annealing temperature is 5◦C below the primer Tm.

Completed reactions can be kept up to 1 week at 4◦C without degradation or longer at–20◦C. In the latter case, minimize freeze-thaw cycles.

4. Clean up reactions using a GenElute PCR Clean-Up Kit according to manufacturer’sinstructions.

Determine if mutations are present using bioanalyzer5. Determine concentration of eluted DNA using a NanoDrop spectrophotometer. Di-

lute all samples to below 50 ng/μl using water.

The Agilent DNA 1000 chips will clog if DNA is run at a concentration >50 ng/μl.

6. Load samples onto a chip from the Agilent DNA 1000 Kit and run chip immediatelyon the bioanalyzer.

7. If mutations are present, proceed to subcloning and sequence confirmation (see BasicProtocol 4).

ALTERNATEPROTOCOL

CLONE IDENTIFICATION: FLUORESCENCE-BASED CAPILLARYSEQUENCING

This protocol describes a method for analyzing single-stranded product. This methodwill not only show the presence or absence of wild-type sequence, it will determine thenumber and size of mutations. This allows the user to funnel only clones with out-of-frame mutations and loss of wild type through to confirmation by sequencing.

Additional Materials (also see Basic Protocol 3)

Nested primers for target genes labeled with 6-carboxyfluorescein (FAM) andhexachloro-6-carboxyfluorescein (HEX)

MicroAmp 96-well reaction plate with rubber septaPowerPlex 16 System Kit (Promega DC6531), including allelic ladder mix and

internal lane standard 600Hi-Di formamideAluma Seal II film3730xl Genetic Analyzer (Applied Biosystems)Peak Scanner software (Applied Biosystems)

Amplify genomic DNA using Cel1 primers1. Amplify genomic DNA as described for bioanalysis (see Basic Protocol 3, steps 1-3).

2. Check DNA concentration and quality on a NanoDrop spectrophotometer.

PCR amplify product using fluorescently labeled nesting primers3. Dilute forward and reverse labeled nested ZFN primers with water to 20 μM upon

receipt to make working stocks. Dilute Cel1 PCR product with water 100×.

4. Prepare PCR reaction as follows:

20 μl JumpStart reaction mix1 μl each labeled primer (forward and reverse)14 μl water4 μl diluted PCR product.

5. Run PCR using the same thermocycler conditions.


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6. Check DNA concentration and quality on the NanoDrop spectrophotometer.

If not used the same day, the product can be kept frozen at –20◦C for up to 1 week.

Determine if mutations are present using capillary sequencer7. Dilute PCR product with water 100×.

8. Add the following to the first well of a MicroAmp plate:

1 μl allelic ladder mix0.5 μl internal lane standard 6009.5 μl Hi-Di formamide.

9. In the required number of wells, prepare samples for sequencing as follows:

1 μl PCR product0.5 μl internal lane standard 6009.5 μl Hi-Di formamide.

10. Include minimum of three wells for a wild-type control.

11. Add 20 μl water to all empty wells of the plate and seal with Aluma Seal II.

0

4000

8000

12000

16000

20000

24000

MDR1 wild-type sequence

257 259 261

263.01 bp

263 265 267

A

0

2000

4000

8000

6000

12000

10000

16000

14000

MDR1 knockout clone sequence

250 252 254 256 258 260 262 264

249.91 bp 259 bp 261.99 bp

B

Figure 23.2.5 Fluorescence-based capillary sequence analysis of MDR1 wild type and an MDR1knockout clone analyzed using ABI Peak Scanner software. (A) The wild-type sequence is 263.01bp. (B) Three knockout clone sequences are identified at 261.99 bp (a 1-bp deletion), 259.00 bp(4-bp deletion), and 249.91 bp (13-bp deletion).

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12. Denature for 3 min at 95◦C in the thermocycler, then immediately chill for 3 min oncrushed ice or in an ice-water bath.

Proceed immediately to analysis. Do not store after denaturing the samples.

13. Remove Aluma Seal and cover wells with rubber septa. Put plate in plate base andcover with plate retainer, clicking to shut. Run on capillary sequencer.

14. Analyze data using Peak Scanner software.

a. Average the wild-type control wells and determine standard deviation (should be<0.10).

b. Determine the peak sizes for each sample and compare to wild type.

c. If no wild-type sequence is present in a sample, subtract the size of the peaks fromthe average wild-type to determine the size of the mutations.

The relative peak heights can be used to determine the number of mutated alleles. Forexample, in Figure 23.2.5B, the peak at 261.99 is close to twice the height of the othertwo peaks. Sequence confirmation shows that this corresponds to two alleles out of fourhaving the 1-bp deletion, while one allele carries the 4-bp deletion and the last carriesthe 13-bp deletion.

15. For samples that contain no wild type and no in-frame mutations, proceed to sub-cloning and confirmation by sequencing (see Basic Protocol 4).

Fluorescence-based capillary sequencing data for the MDR1 alleles from theMDR1/BCRP double-knockout cell line are shown in Figure 23.2.5.

BASICPROTOCOL 4

CONFIRMATION OF CLONE IDENTIFICATION: TA CLONING AND GENESEQUENCING

This protocol describes the process used to prepare clones for gene sequencing. GenomicDNA is amplified using Taq polymerase, combined with TOPO vector, and transformedinto competent cells. The cells are grown on agar containing antibiotic to select forcolonies with the insert. This process allows for each allele of the gene of interest to beanalyzed separately.

Materials

PCR-amplified DNA from clone(s) of interest (see Basic Protocol 3 or AlternateProtocol)

TOPO TA Cloning Kit for Sequencing with One Shot Top10 ChemicallyCompetent E. coli (Invitrogen K4575), including vector, salt solution, and SOCmedium

Selective plates: 100-mm LB agar plates with 100 μg/ml carbenicillin42◦C water bath

Additional reagents and equipment for transforming cells (APPENDIX 3A)

1. Determine the appropriate amount of PCR product to use for vector ligation basedon a 1:3 vector/insert ratio, then pre-dilute PCR product with water. Set up the TOPOcloning reaction as follows (6 μl total):

0.5-4 μl PCR productwater to 4 μl1 μl salt solution1 μl of 10 ng/μl TOPO vector.

Make sure to add TOPO vector last, as it is salt sensitive.


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BCRP Wild-TypeTACACCACCTCCTTCTGTcatcaACTCAGATGGGT

BCRP Alleles 1 & 2: 5-bp deletionTACACCACCTCCTTCTGTcatcaACTCAGATGGGT

BCRP Allele 3: 16-bp deletionTACACCACCTCCTTCTGTcatcaACTCAGATGGGT

BCRP Allele 4: 20-bp deletionTACACCACCTCCTTCTGTcatcaACTCAGATGGGT

MDR1 Wild-TypeGTCCTGTTCTTGGACtgtcaGCTGTGTCTGGGCAAAG

MDR1 Alleles 1 & 2: 1-bp deletionGTCCTGTTCTTGGACtgtcaGCTGTGTCTGGGCAAAG

MDR1 Allele 3: 4-bp deletionGTCCTGTTCTTGGACtgtcaGCTGTGTCTGGGCAAAG

MDR1 Allele 4: 13-bp deletion � 1-bp substitutionGTCCTGCTCTTGGACtgtcaGCTGTGTCTGGGCAAAG

Figure 23.2.6 Genotype of MDR1/BCRP knockout clone. The entire ZFN binding site is shown.Lowercase, ZFN cut site; bold, deletions; bold/underline, substitution.

2. Mix gently and incubate 5-30 min at room temperature. Place reaction on ice.

3. While reaction is incubating, warm SOC medium to room temperature and place1 vial of competent cells for each transformation on ice to thaw.

It is imperative to thaw competent cells on ice. If cells are warmed too quickly or warmedmultiple times, they will not take up insert.

4. Add 2 μl TOPO reaction to 1 vial thawed competent cells and mix gently. Incubateon ice 5-30 min.

Do not mix by pipetting, as the cells are sensitive and will shear.

The length of incubation here and in step 2 will affect the number of final colonies. Thelonger the incubation time, the more colonies will form.

5. While cells are incubating, label two selective plates for each reaction and warm ina 37◦C incubator.

6. Heat shock cells in a 42◦C water bath for 30 sec without shaking and transfer backto ice.

7. Add 250 μl room temperature SOC medium, cap tube tightly, and place in 37◦Cshaking incubator for 1 hr.

8. Spread 10-50 μl from each transformation on prewarmed selective plates and incu-bate at 37◦C overnight.

9. Pick colonies, isolate DNA by miniprep, and submit for sequencing.

The genotype determined from sequencing data for the MDR1/BCRP double knockout isshown in Figure 23.2.6.

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BASICPROTOCOL 5

VALIDATION OF DRUG TRANSPORTER KNOCKOUT CELL LINE BYTRANSWELL ASSAY

This protocol is designed to assess drug transporter functionality in C2BBe1 cells. Theexperiment must include both the genetically modified C2BBe1 knockout cells andwild-type C2BBe1 cells. Transport is measured in both directions (apical-to-basal andbasal-to-apical) across the cell monolayer, enabling an efflux ratio to be determined. Itis expected that the efflux ratio from the knockout cells will be significantly lower thanthat from wild-type cells.

In this study, buffer is taken from the receiver compartment after a designated time point.Compound concentrations in the receiver samples are quantified by LC-MS/MS, andthe apparent permeability coefficient (Papp) and efflux ratio of the compound across themonolayer are calculated.

Materials

C2BBe1 cells, knockout and wild type, each growing in T-75 flasks and harvestedat 90-100% confluency

0.25% trypsin/EDTA10% 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 acetonitrileHPLC mobile phase A: 4 mM ammonium formateHPLC mobile phase B: 4 mM ammonium formate in 90% (v/v) acetonitrile

50-ml Falcon tubes24-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.

Seed C2BBe1 cells on Transwell inserts1. Aspirate the medium from two T-75 flasks of C2BBe1 cells (one for knockout, one

for wild-type control).

2. Add 3 ml of 0.25% trypsin/EDTA and incubate for 5-7 min at 37◦C.

3. Transfer the cell suspension into a 50-ml Falcon tube, then centrifuge for 5 min at800 rpm to pellet the cells.

4. Aspirate the supernatant, add 10 ml of 10% growth medium to the pellet, andresuspend the cells.

5. Count cells using a hemacytometer (APPENDIX 3B), then adjust to a density of 8 × 104

cells/ml. Seed 500 μl cells into 24-Transwell plates (4 × 104 cells/insert).

6. Add 35 ml of 10% growth medium to the bottom plate (feeder tray).

7. Change the medium twice a week until assay day (21 days).

Perform Transwell assay8. Aspirate medium from the insert and feeder tray and replace with buffer B (500 μl

in the insert and 35 ml in the feeder tray). Incubate at 37◦C for 15 min.

9. Replace the feeder tray with a 24-well plate and aspirate all buffer B. Depending onthe study design, add test compound working solution to the apical (500 μl) or basal


Finger Nucleases

23.2.16


(1000 μl) wells, and add buffer B to the other (basal or apical) wells. Incubate at37◦C for 2 hr.

10. Take 250 μl from the appropriate wells, depending on the direction of transport (i.e.,from the basal well for A-to-B transport, or the apical well for B-to-A transport).Transfer to a 96-well plate and add 10 μl of 25 μM internal standard solution. Sealthe plate with film.

At this point, one may proceed immediately to LC-MS/MS or store the plate up to 4 weeksat –20◦C.

Perform LC-MS/MS11. Analyze C2BBe1 permeates and start solutions by LC-MS/MS using the conditions

described below. Generate peak area data for analytes and the internal standard aswell as peak area ratio (PAR) data (analyte/internal standard).

Substrate solutions may be susceptible to nonspecific binding and thus, if not frozen,should be analyzed immediately.

LC-MS/MS is a rapid, sensitive, and specific approach to measurement of substrateconcentration in the Transwell solutions (Wang et al., 2000).

a. HPLC conditions:

Instrument: Agilent 1100Software: Agilent ChemStationColumn: Agilent Zorbax SB-C8, 2.1 × 50 mm, 3.5 μmColumn temp: AmbientSample temp: 4◦-12◦CInjection volume: 40 μlFlow rate: 0.5 ml/min (flow divert 0-0.5 min)Mobile phase A: 4 mM ammonium formateMobile phase B: 4 mM ammonium formate in 90% (v/v) acetonitrileGradient: See Table 23.2.1.

b. General MS conditions:

Instrument: ABI 4000 QTrapSoftware: Analyst 1.5Source: Turbo sprayCurtain gas: 10Source temp: 550Gas 1: 25Gas 2: 50Interface heater: OnIS voltage: 4500.00 (+ or −)CAD cas: MediumQ1 resolution: UnitQ3 resolution: Unit.

c. Multiple reaction monitoring (MRM): Create MRM experiments with analyte andinternal standard parent-product ion pairs.

Specific MRM settings are instrument-specific and may need to be varied. Refer toTable 23.2.2 below.

12. Calculate peak area ratio (PAR) as the ratio of analyte peak area to internal standardpeak area.

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Table 23.2.1 HPLC gradient for LC-MS/MS

Time (min) %A %B

1 90 10

2 1.0 40 60

3 1.8 40 60

4 2.0 5 95

5 3.0 5 95

6 3.5 90 10

7 4.0 90 10

Table 23.2.2 Recommended Analyte-Specific MRM Settings

Analyte Polarity Q1 Q3 DP CE CXP

Tolbutamide (IS) + 271.2 172.4 60 18 25

Tolbutamide (IS) − 269.1 170.2 −76 −25 −13

Digoxinb + 798.6 651.6 60 18 25

Metoprolol + 268.3 116.1 60 26 20

Estrone-3-sulfate − 349.1 269.1 −118 −44 −10

Actinomycin D + 1256.6 300.3 160 76 15aIS, internal standard.bDigoxin parent ion is [M+NH4]+.

Determine Papp value and efflux ratio13. Calculate the permeability coefficient as follows;

PA C

dMdtapp

r

0

=1

where A is area (cm2), C0 is mass of compound initially in the donor compartment,and dMr/dt is the rate of drug permeation across the cells.

14. Calculate the efflux ratio (ER) as the ratio of Papp determined in the A→B directionto Papp determined in the B→A direction:

ER app,B A app,A B= P P→ →

BASICPROTOCOL 6

MEASUREMENT OF CELL MONOLAYER INTEGRITY USING LUCIFERYELLOW

Evaluation of permeability characteristics of C2BBe1 cells can be performed by mea-suring passive passage of different molecules across the monolayer. Small hydrophiliccompounds cross the monolayer mainly via the paracellular space, such as through thetight junctions, and can be considered markers of passage by this route (UNIT 20.6). Luciferyellow is one such marker that is easily detectable. It is used to check the barrier integrityand to determine whether the working concentration of a test compound disturbs theintegrity of the monolayer. In this protocol, the Lucifer yellow assay is conducted afterthe Transwell assay.


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Materials

Transwell assay samples (see Basic Protocol 5)Buffer B (see recipe)0.1 mg/ml Lucifer yellow (CH dipotassium salt, Sigma L0144) in buffer B

96-well plateSpectrometer

1. After removing samples for LC-MS/MS (see Basic Protocol 5, step 10), aspirate theremaining liquid from the apical wells.

2. Add 500 μl of 0.1 mg/ml Lucifer yellow to the apical wells and 1000 μl buffer B tothe basal wells.

3. Incubate at 37◦C for 60 min.

4. Transfer 150 μl from the basal wells to a 96-well plate and read in a spectrometerwith excitation at 485 nm and emission at 535 nm. Also measure absorbance forbuffer B (blank) and 0.1 mg/ml Lucifer yellow.

5. Calculate the percent permeability from the absorbance values as follows:

% permeability =sample blank

Lucifer yellow blank

−−

⎡

⎣⎢ ⎡

⎣⎢

×100

A permeability of <3% is acceptable.

REAGENTS AND SOLUTIONSUse Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stocksolutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Buffer B

500 ml HBSS (APPENDIX 2A) containing:12.5 ml 1 M D-glucose10 ml 1 M HEPES biological buffer1 ml 625 mM CaCl21 ml 250 mM MgCl2Adjust to pH 7.4Store up to 4 weeks at 4◦C

Growth medium, 10% or 20%

Dulbecco’s modified Eagle medium (DMEM) containing:10% or 20% heat-inactivated fetal bovine serum (FBS)1% penicillin/streptomycin (final 100 U penicillin, 0.1 mg streptomycin)2 mM L-glutamineStore up to 4 weeks at 4◦C

Sorting buffer

HBSS (APPENDIX 2A) containing:1% bovine serum albumin (BSA, fraction V)1 mM EDTA25 mM HEPESMix well to dissolve BSA completelyPass through a 0.2-μm filterStore up to 4 weeks at 4◦C

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Test compound working solution

Stock solution: Dissolve compound in DMSO and vortex to mix. If necessary, warmor sonicate to dissolve completely. Store up to 6 months at 4◦C

Working solution: Dilute stock 1:200 with HBSS to make a working solution witha final DMSO concentration of 0.5% (v/v). Prepare fresh just before use.

COMMENTARY

Background InformationAlthough transporter assays and their inter-

pretation have improved dramatically in recentyears, the translation of in vitro to in vivo andpre-clinical to clinical drug transport data isstill very challenging. There is currently a re-newed focus on transporters, as evidenced bythe 2010 White Paper published by the Interna-tional Transporter Consortium and sponsoredby FDA Critical Path (International Trans-porter Consortium, 2010). There are numerousclinical examples of drug failure due to un-expected interaction with transporters. Someof the current hurdles for assessing whethera compound is a substrate and/or inhibitor ofthe key transporters are that (1) transportersare overexpressed in non-relevant cell lines,(2) most prototypical inhibitors/substrates arenot selective, and (3) animal models differin transporter expression and function whencompared with human. To address these hur-dles, we describe a method for creating mod-ified cells lines in which specific transportersare knocked out in a human cell line.

Methods for studying transporter activitycan be divided into two broad assay systems:membrane-based assay systems and cell-basedassay systems. The protocols described hereinfocus on a cell-based assay system that willidentify the specific transporter(s) involved indrug-drug interactions of potential drug can-didates. The protocol for creating the modi-fied cells lines is based on zinc finger nucle-ase (ZFN) technology (licensed by Sigma LifeSciences). ZFNs are engineered DNA-bindingproteins that facilitate targeted editing of thegenome by creating double-stranded breaksin DNA at specified locations. The double-stranded breaks are important for site-specificmutagenesis in that they stimulate the cell’snatural DNA-repair processes, namely homol-ogous recombination and non-homologousend joining (NHEJ). These cellular processescan be harnessed to generate precisely targetedgenomic edits resulting in cell lines with tar-geted gene deletions (knockouts).

The discovery and manipulation of zinc fin-ger protein (ZFP) domains started a field of

research that enables a very targeted approachto genetic manipulation in higher-level eukary-otes. This research culminated in the forma-tion of engineered ZFNs. To date, ZFNs havebeen used successfully to create single, dou-ble, triple, and tetraploid gene knockouts incells, targeted knockouts in animals, and tar-geted gene insertions in native chromosomalloci. This particular approach to gene targeting(a genetic technique that uses homologous re-combination to change an endogenous gene)was chosen for this study for the followingreasons. (1) Other methods, which rely ondriving homologous recombination throughexogenously supplied donor DNA and thespecified locus, demonstrate low efficiency re-combination, which necessitates sophisticatedpositive and negative selection strategies toisolate the rare targeted events. (2) The ZFNapproach enables a very targeted and specificapproach through the specific cleavage do-main of the Fok1 endonuclease and the target-ing power of the zinc finger protein. (3) Ge-nomic manipulation using ZFNs is 1000 timesmore efficient than the prevailing homologousrecombination−based mechanism, eliminat-ing the need for selection techniques. (4) Thechallenge of ploidy is overcome through theuse of ZFNs, as demonstrated by the knockoutdescribed in this unit, which modified eightalleles in the MDR1/BCRP double-knockoutcell line. The limitations associated with genetargeting through the use of ZFN technologyare the tremendous amount of expertise andresources that are required for application ofthis technology.

Critical Parameters andTroubleshooting

Validation of knockout in C2BBe1 cellsEvaluation of drug transporter function-

ality using bidirectional Transwell assays inC2BBe1 knockout cells is critical to con-firm the knockout effect. Currently, thereis no universally accepted standard protocolfor conducting cell-based bidirectional Tran-swell assays. Standardization of experimental


Finger Nucleases

23.2.20


conditions such as assay pH, drug concentra-tion, duration of incubation, and duration ofculture (usually 3 weeks) can provide betterconsistency of the results and significantly re-duce false negatives (i.e., unable to identify atrue substrate). The use of an optimal substrateconcentration is key for attaining accurate re-sults (Balimane et al., 2006).

The integrity of the cell membrane barrieris an essential element for transport experi-ments. Data can only be used if the C2BBe1monolayer is undamaged during culture andis not affected by the test compound. Cellscultured in Transwell devices can introduce alot of inconsistency and variation in the re-sults; passage number, cell growth rates, andinappropriate experimental methods may con-tribute to the variation. In addition to Luciferyellow, several other methods can be usedto verify the integrity of the cell membrane,including passage of mannitol and measure-ment of transepithelial electrical resistance(UNIT 20.6).

The efflux ratio of substrates is depen-dent on the number of cell passages. A rela-tively long cycle time for the clone generation/selection process results in a higher cell pas-sage number from knockout cell lines pro-duced. Thus, for controls, it is necessary to usewild-type cells that have an equivalent passagenumber to the knockout cells.

Several efflux transporters have been suc-cessfully knocked out in C2BBe1 cells. TheMDR1 single- and MDR1l/BCRP double-knockout cell lines were tested with theMDR1 substrate digoxin. BCRP single andMDR1/BCRP double-knockout cells lines

were tested with the BCRP substrate estrone-3-sulfate. The efflux ratio of digoxin was dra-matically reduced in MDR1 knockout cells,while the other substrates were not signif-icantly affected. The MDR1/BCRP double-knockout shows a significant reduction intransport of both digoxin (MDR1) and estrone-3-sulfate (BCRP). The efflux ratio of estrone-3-sulfate from the BCRP knockout cell linewas dramatically reduced (Fig. 23.2.7). MRP2activity in these knockout cell lines was alsoevaluated using the MRP2 substrate actino-mycin D. All knockout cell lines retain MRP2functionality, although the efflux ratio of acti-nomycin D showed relatively high variationamong knockout cell lines due to high nonspe-cific binding. Based on these data, these newlydeveloped drug transporter knockout cell linesprovide novel tools for identifying substratesand inhibitors of drug transporters.

Sensitivity of LC-MS/MSIt is important to verify that the LC-MS/MS

method is capable of detecting/quantifying an-alytes at the following concentrations: 50 nMdigoxin, 500 nM metoprolol, 50 nM estrone-3-sulfate, and 100 nM actinomycin D. It isrecommended that quality control samples beprepared and analyzed just prior to test sam-ples to verify adequate system performance.

Use of different cell typesThere are a number of critical technical

considerations that must be evaluated in thecell type that you are going to modify beforeyou begin the genome editing process. Param-eters to consider are: ZFN functional activity,

0

40

20

60

80

100

120

140

Eff

lux

rati

o (

B t

o A

/A t

o B

)

Substrate (10 �M)

Efflux Ratio of Substrates

Estrone-3-sulfate Actinomycin-DDigoxin Metoprolol

BCRP KO

MDR1 KO

MDR1/BCRP KO

WT

Figure 23.2.7 Efflux ratio of MDR1 substrate digoxin, MRP2 substrate actinomycin D, and BCRPsubstrate estrone-3-sulfate from a Transwell assay. Negative control is metoprolol.

DrugTransporters

23.2.21


delivery of ZFNs, and growth conditions forsingle-cell cloning (Woodcock et al., 1991;Urnov et al., 2010; Collin and Lako, 2011).

The first parameter to establish is the func-tional activity of the ZFNs in the particularcell line chosen for modification. A minimumacceptable cutting efficiency for a ZFN pair is>1%; however, if the cell line is tetraploid (asis the cases for C2BBe1 cells), a higher cuttingefficiency is imperative. The functional activ-ities of ZFNs have been improved through thedesign of new interdomain linkers, the devel-opment of heterodimeric variants of the FokIdimerization interface, and the use of mild hy-pothermia (Miller et al., 2007; Szczepek et al.,2007; Handel et al., 2009; Doyon et al., 2010).

Delivery of ZFNs can be achieved bycalcium phosphate transfection, lipofection,electroporation, nucleofection, viral delivery,or direct injection; however, nucleofection andelectroporation generally produce the highestDNA cleavage efficiencies. ZFNs are suppliedas either DNA or mRNA transcripts, but de-livery of ZFNs as mRNA transcripts results indecreased cytotoxicity, diminished off-targetevents, and higher efficiency. Additionally, us-ing the mRNA transcript eliminates the riskof random integration, the need to test differ-ent promoters, and the necessity for nucleardelivery.

The third technical consideration is the abil-ity of the cell type to go through the single-cell cloning process. Some of the conditions toconsider are dilution cloning versus FACs sort-ing, percentage of serum and/or other growthfactors, and the use of conditioned media,coated tissue culture plates, and feeder cells.

Anticipated ResultsThe cutting efficiency of the ZFNs used for

genome editing is critical. When ZFNs are pur-chased from Sigma-Aldrich, documentation ofthe cutting efficiency from the Cel 1 assayis provided in the certificate of analysis. Thenumber of single cell clones you will need toscreen to find a homozygous diploid knockoutdepend on the cutting efficiency. As a generalrule, if the cutting efficiency is 1-3% based onCel 1 activity, you should screen more than300 clones or use selection methods. If cuttingefficiency is >3%, screen at least 100 clones.Recalling that C2BBe1 cells are tetraploid fortransporter genes, we recommend using ZFNswith >6% cutting efficiency and screening aminimum of 300 clones to find at least onetetraploid knockout.

Clonal growth following single-cell sort-ing is also an important number to have de-termined. C2BBe1 cells have a clonal growout rate of ∼20% (number of cells that formcolonies post sorting divided by the total num-ber of wells sorted). Using that percentageto generate 300 clones to screen, one shouldsingle-cell sort fifteen 96-well plates. Whenmodifying more that one gene by genome edit-ing, the number of clones that must be screenedincreases dramatically.

Time ConsiderationsThe entire process for generation of trans-

porter knockout cell lines by ZFN technologyis shown in Figure 23.2.2. It is divided intothree sections: clone generation, clone iden-tification, and functional analysis. The clonegeneration process takes the longest to com-plete, with an approximate time of 14 weeks.Clone identification and confirmation of geno-types takes approximately 2 weeks. Func-tional analysis, including expanding the cellsand testing them in the 21-day Transwell as-say, takes approximately 5 weeks. In total, tocomplete the entire process will take approxi-mately 21 weeks.

Literature CitedBalimane, P.V., Han, Y., and Chong, S. 2006. Cur-

rent industrial practices of assessing perme-ability 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 forstudies of drug transport across human intestinalepithelium. In Cell Culture Models of BiologicalBarriers (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 fingernuclease-driven targeted genetic editing in hu-man 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. Herita-ble targeted gene disruption in zebrafish usingdesigned 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 coldshock enhances zinc-finger nuclease-mediatedgene disruption. Nat. Methods 7:459-460.

Handel, E.M., Alwin, S., and Cathomen, T. 2009.Expanding or restricting the target site reper-toire of zinc-finger nucleases: The inter-domainlinker as a major determinant of target site se-lectivity. Mol. Ther. 17:104-111.


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International Transporter Consortium. 2010. Mem-brane 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., Gre-gory, P.D., Pabo, C.O., and Rebar, E.J. 2007. Animproved zinc-finger nuclease architecture forhighly specific genome editing. Nat. Biotechnol.25:778-785.

Peterson, M.D. and Mooseker, M.S. 1992. Char-acterization of the enterocyte-like brush bordercytoskeleton of the C2BBe clones of the humanintestinal 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 genomemodification in the crop species Zea maysusing 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 re-duces 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 editingwith 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 permeability ofdrug candidates through a Caco-2 cell mono-layer by liquid chromatography/tandem massspectrometry. J. Mass Spectrom. 35:71-76.

Woodcock, S., Williamson, J., Hassan, I., andMackay, M. 1991. Isolation and characterizationof clones from the Caco-2 cell line displayingincreased taurocholic acid transport. J. Cell Sci.98:323-332.

Key Resourcehttp://www.sigmaaldrich.com/life-science/zinc-

finger-nuclease-technology.htmlWebsite for technical help with targeted genomeediting using zinc finger nucleases.

Documents

Current Protocols in Toxicology Volume 51 || Use of Zinc Finger Nuclease Technology to Knock Out Efflux Transporters in C2BBe1 Cells