Mitosis is a dynamic and stochastic process that has evolved to accurately segregate the
genetic material into two daughter cells, thereby preventing genomic instability and the
development of disease. Mitosis is a phenomenally complicated process that involves hun-
dreds, if not thousands, of protein components and regulatory steps that operate in both
space and time to drive cell division. Modern methods make it possible to ask for mechanis-
tic principles underlying this bewildering complexity. The main purpose of this volume is
to provide an up-to-date collection of methods and approaches that are used to investigate
the mechanism of mitosis at the molecular level. While many of these methods are focused
on mammalian cells, we have, where appropriate, included chapters using model organ-
isms. We hope to capture both current approaches and the future direction of method
development, with contributions from both established researchers and emerging young
This book is designed with two general groups of readers in mind: First, graduate stu-
dents and postdoctoral researchers who are beginning work for the first time in a mitosis
laboratory. Second, researchers who are already working in the mitosis field who require
a resource for both established and newly developed methodologies. To achieve this,
the organization of this book developed into three general areas: First, we cover meth-
ods that can be used to inactivate your gene of interest, or deplete proteins of interest
(chapters 1–3). Second, we learn about specific biochemical and microscope-based meth-
ods (chapters 4–9). Third, we discover approaches to monitor and measure key mitotic
processes (chapters 9–20). Given the complexities of mitosis, it seems highly probable that
such a combination of imaging, biochemical and genetic methodologies will be crucial to
our future understanding of mitotic regulation.
I would like to thank all of the authors for their enthusiasm and effort in putting
together this set of methods, as well as all the members of my laboratory for proof reading
and correcting the chapters and especially Sarah McClelland who read all the chapters
at least twice at various stages. Finally, I wish to thank Lyndy Rasmusen for her help in
administering the project and keeping me organized.
Andrew D. McAinsh
Functional Dissection of Mitotic Regulators Through Gene
Targeting in Human Somatic Cells
Eli Berdougo, Marie-Emilie Terret and Prasad V. Jallepalli
With the human genome fully sequenced (1, 2), biologists continue to face the challenging task of
evaluating the function of each of the ∼25,000 genes contained within it. Gene targeting in human
cells provides a powerful and unique experimental tool in this regard (3–8). Although somewhat more
involved than RNAi or pharmacological approaches, somatic cell gene targeting is a precise technique
that avoids both incomplete knockdown and off-target effects, but is still much quicker than analogous
manipulations in the mouse. Moreover, immortal knockout cell lines provide excellent platforms for both
complementation analysis and biochemical purification of multiprotein complexes in native form. Here
we present a detailed gene-targeting protocol that was recently applied to the mitotic regulator Polo-like
kinase 1 (Plk1) (9).
Key words: Gene targeting, rAAV, human somatic cells, mitosis, Plk1.
Over the past several years, the methods used to generate knock-
out or knockin mutations in human somatic cells have greatly
improved in efficiency (3–8). The current method utilizes the
high recombination potential of adeno-associated virus (AAV)
vectors, requires less than 6 months for homozygous mutation
of both alleles in a diploid cell (10–12), and can be divided into
several general stages. The first step is to design and assemble a
targeting vector containing 5’ and 3’ arms that are homologous
to the locus of interest. If desired, gene inactivation can be made
conditional, simply by placing loxP sites on either side of a coding
Andrew D. McAinsh (ed.), Mitosis: Methods and Protocols, vol. 545
© Humana Press, a part of Springer Science+Business Media, LLC 2009
DOI 10.1007/978-1-60327-993-2 2, Springerprotocols.com
22 Berdougo, Terret, and Jallepalli
exon of interest. This allows the exon to be rapidly and irreversibly
deleted at the desired time, via expression of Cre recombinase.
This approach permits the study of genes that are essential for cell
proliferation, as expected for many mitotic regulators. The second
step is to cotransfect the targeting vector with helper plasmids into
HEK293 cells, in order to generate infectious AAV particles. The
third step is to infect the cell line of interest with these viruses
and subsequently screen for clones that have undergone correct
locus-specific recombination. The final step is to verify gene dis-
ruption by PCR and Southern blotting assays, and finally, to carry
out the procedure a second time to obtain homozygous mutant
Multiple human cell types are amenable to gene target-
ing with AAV vectors, including both transformed and non-
transformed cell lines and primary cells isolated from patients
(7, 9, 13). Here we use telomerase-immortalized human reti-
nal pigment epithelial cells (hTERT-RPE) to investigate Plk1
function in a nontransformed, nontumorigenic setting (9).
Unlike established cancer cell lines, hTERT-RPE cells toler-
ate severe (>90%) depletion of Plk1 via RNAi without effect
(14). In contrast, we find that homozygous deletion of the
PLK1 locus fully abrogates its function and recapitulates all
known mitotic functions of this kinase (9). Furthermore, these
PLK1?/?cells could be reconstituted with a variant form of Plk1
that is uniquely susceptible to bulky ATP analogues, enabling
chemical genetic dissection of Plk1’s roles in late mitosis and
2.1. Cell Culture
1. HEK293 cells (ATCC CRL-1573)
2. Telomerase-immortalized human retinal pigment epithelial
cells (hTERT-RPE; ATCC CRL-4000)
3. Medium suitable for propagation of HEK293 cells:
D-MEM (Invitrogen) supplemented with 10% fetal bovine
serum (FBS; Omega Scientific) and 0.1 mg/ml penicillin-
streptomycin (Gemini Bio-Products)
4. Medium suitable for propagation of hTERT-RPE cells:
D-MEM/F:12 medium with 15 mM HEPES, 2.5 mM
L-glutamine, 2.4 g/L sodium bicarbonate (Invitrogen),
supplemented with 10% FBS and 0.1 mg/ml penicillin-
5. Hank’s balanced salt solution (HBSS; Invitrogen)
6. 0.05% Trypsin-EDTA (Invitrogen)
Gene Targeting in Human Cells 23
7. Tissue culture-treated 96-, 48-, 24-, 12-, and 6-well plates
8. Tissue culture treated T-25 and T-75 flasks (Corning)
9. G418 (Gemini Bio-Products)
10. Presterilized 8-port manifolds (CLPdirect)
11. Repeater Plus pipetter with sterile 50-ml Combitips
12. 100 ml sterile basins (Fisher)
P200 and filtertips
2.2. Production of
rAAV Particles and
1. pNY, Jallepalli lab (9)
2. QuikChange II XL site-directed mutagenesis kit (Strata-
3. AAV Helper-free system (Stratagene, 240071)
4. Lipofectamine Transfection reagent/Plus reagent (Invitro-
5. OptiMEM I medium (Invitrogen)
6. Disposable cell scrapers (Fisher)
7. Deep-well blocks (BD Biosciences)
8. WizardSV 96 genomic
9. Beckman-Coulter Allegra 25R with deep-well block rotor
(S5700) or Vac-Man 96 Vacuum Manifold (Promega
10. Multichannel P200 and P1000 pipetters and filter tips
11. QIAampDNA Blood Mini Kit (Qiagen, 51106)
2.3. PCR Screening
1. Thin-wall 96-well plates (Simport) and sealing films
2. Tissue culture-grade water (Sigma)
3. DMSO (Sigma)
4. 10X PCR buffer: 166 mM ammonium sulfate, 670 mM
Tris-HCl, pH 8.8, 67 mM MgCl2, 100 mM beta-
5. 10 mM dNTPs (USB)
6. Primers (Integrated DNA Technologies)
7. Platinum Taq polymerase (Invitrogen)
8. Taq Extender (Stratagene)
9. Mineral oil (Sigma)
10. Bio-Rad Sub-Cell Model 96/192 gel system
11. Multichannel P10, P20, P200 pipetters and filter tips
Excision of the Neo
1. Fugene 6 (Roche)
2. pCAGGS-FLPe (Gene Bridges)
3. Puromycin (Gemini Bio-Products)
4. Purified adenovirus expressing Cre recombinase (Vector
Development Laboratory Baylor College of Medicine)
24 Berdougo, Terret, and Jallepalli
The following method can be used to generate a conditional
knockout of any desired locus in human somatic cells. The tar-
geting strategy should be planned in its entirety before starting
work (Fig. 2.1).
Fig. 2.1. Timeline for generating a conditional knockout in human somatic cells.
of the Genetic Locus
and Primer Design
1. Choose the exon that you wish to conditionally delete. This
exon will need to be flanked by tandemly oriented loxP sites,
generating a so-called “floxed” allele. Use Ensembl to ver-
ify that length of this exon in basepairs is not a multiple
of three (http://www.ensembl.org/index.html). This will
ensure that the open reading frame undergoes a frameshift
and premature termination after the exon is deleted.
2. Using RepeatMasker (http://www.repeatmasker.org/cgi-
bin/WEBRepeatMasker), examine a 4-kb region centered
on this exon for repetitive DNA content.
3. Choose 5’ and 3’ homology arms of about 1.0–1.5 kb in
length, taking care to maximize the unique sequence content
in each arm (see Fig. 2.2A and Notes 1 and 2).
4. Design oligonucleotides for amplifying and cloning the
homology arms into the shuttle vector pNY, which contains
a central FRT-neoR-FRT-loxP cassette (see Fig. 2.2A, primer
pairs F1/R1 and F2/R2; see Notes 3 and 4).
5. Design several oligonucleotides for PCR screening and
sequencing across both homology arms. The candidate
Gene Targeting in Human Cells 25
Fig. 2.2. Strategy for targeting exon 3 of PLK1. (A) Schematic of the 5’ end of the PLK1 locus. Primers used to amplify
the homology arms for cloning are denoted by black arrows, and those used for PCR screening are denoted by white
arrows. (B) Map of the pNY polylinker. FRT sites are shown as white circles and the loxP site is shown as a triangle.
Unique restriction sites for cloning the 5’ and 3’ homology arms are also indicated. (C) Structure of the pNY-PLK1floxand
pNY-PLK1Δshuttle plasmids. (D) Transfer of the NotI fragment from pNY-PLK1floxto pAAV generates the final construct
(pAAV-PLK1flox) used for virus production. ITR, AAV-specific inverted tandem repeats.
screening primers should correspond to unique genomic
sequences about 100–500 bp outside the region delimited
by the 5’ and 3’ homology arms (see Fig. 2.2A screening
primers F3, F4, and R3).
6. Harvest wild-type RPE-hTERT cells from a T-25 flask and
prepare genomic DNA (gDNA) using the QIAamp DNA
Blood Mini Kit. Determine the concentration of your DNA
by measuring the OD 260 of your sample in a spectropho-
7. Perform test PCR reactions using the conditions specified in
Section 3.6 with the candidate screening primers and locus-
specific primers (F3/R1, F4/R1, F2/R3; see Fig. 2.2A),
26 Berdougo, Terret, and Jallepalli
using the gDNA prepared in step 6 as the template. Pre-
pare 10-fold serial dilutions of your gDNA to identify those
primers which have optimal sensitivity and specificity (see
3.2. Construction of
the Targeting Vector
1. Using a high-fidelity thermostable polymerase (e.g., Pfu-
Turbo, Stratagene), amplify 5’ and 3’ homology arms from
human gDNA (e.g., purified from the cell line of interest)
or a human genomic BAC (bacterial artificial chromosome)
clone identified by BLAST searching and obtained commer-
cially (Invitrogen or BACPAC Resources).
2. Subclone homology arms into pNY and verify by sequencing
(see Fig. 2.2B).
3. Introduce a loxP site into the appropriate homology
arm of the targeting vector by site-directed mutagenesis
(QuikChange II XL kit, Stratagene) or linker ligation. This
loxP site should be in the same orientation as the existing
loxP site in pNY. The introduced loxP site should also be
marked with a novel restriction endonuclease site to facili-
tate downstream analyses. Verify the presence, orientation,
and integrity of the inserted elements by sequencing and
restriction digestion (see Note 6).
4. Restriction digest pNY containing the homology arms with
NotI and gel-purify the insert (5’ arm-[FRT-neoR-FRT-loxP
5. Restriction digest the recipient vector pAAV-lacZ with NotI.
Treat this reaction with 1 ?l of Calf Intestinal Phosphatase
(CIP) and leave reaction at 37◦C for an additional 15 min.
Extract the DNA with phenol:chloroform, ethanol precip-
itate, and resuspend in 20 ?l water. Gel-purify the NotI
digested pAAV vector backbone (see Note 7).
6. Ligate the NotI digested insert from step 4 into the pAAV
backbone from step 5 (see Fig. 2.2D).
7. Transform the ligations and screen the resulting colonies
for recombinant AAV plasmids that contain your homology
arms (see Note 8).
8. Prepare transfection-grade plasmid DNA using standard
methods (silica-based kits or isopycnic centrifugation on
cesium chloride gradients).
3.3. Generation of
1. Thaw early-passage HEK293 cells in D-MEM supplemented
with 10% FBS and 0.1 mg/ml penicillin-streptomycin (com-
plete D-MEM). Grow cells at 37◦C in a tissue culture incu-
bator until 80–90% confluent.
2. Split HEK293 cells into two T-75 flasks and grow at 37◦C
in a tissue culture incubator until they are at 50–70%
Gene Targeting in Human Cells27
3. Transfect each flask with 3 ?g each of your targeting vec-
tor, pHELPER, and pAAV-RC (9 ?g total) using Lipofec-
tamine reagent and Plus reagent as follows. Mix DNAs in
one sterile 1.5 ml tube with 750 ?l OptiMEM I reduced
serum medium and 36 ?l of Plus reagent. In a second ster-
ile 1.5 ml tube, mix 54 ?l of Lipofectamine reagent with
750 ?l OptiMEM I reduced serum medium. After 15 min
at room temperature, drip the contents of the first tube into
the second tube, and incubate for 30 min at room temper-
ature to form DNA/lipid complexes. Wash the HEK293
cells twice with Hank’s balanced salt solution (HBSS) and
add 7.5 ml OptiMEM and incubate at 37◦C for 15 min.
Drip this DNA/lipid mixture onto your HEK 293 cells
and incubate for 4 h at 37◦C. Replace the medium with
15 ml of complete D-MEM and return the cells to the 37◦C
4. Harvest the virus particles three days post-transfection.
Begin by transferring the medium from each T-75 to a sterile
50 ml conical tube (see Note 9).
5. Add 5 ml of complete D-MEM to each flask, scrape off the
remaining cells using a disposable cell scraper, and transfer
each cell suspension to the 50 ml conical tube.
6. Set up a dry ice/methanol bath. Freeze the cell suspension in
the bath. Transfer the tubes to a 37◦C water bath and thaw.
When the cell suspension is almost fully thawed, vortex the
tubes at maximum speed for 1 min. Repeat this freeze/thaw
cycle two more times (see Note 10).
7. Spin out the cell debris at 10,000 × g for 30 min at 4◦C.
Decontaminate the outside of each tube with 70% ethanol
and place within a sterile tissue culture hood.
8. Carefully transfer the supernatant fraction to a new 50 ml
conical tube. This is your working stock of infectious rAAV
particles. Aliquot in 5–10 ml fractions and store at –80◦C
until ready to use.
3.4. Infection of
Target Cells with
rAAV Particles and
Selection for Stable
1. Thaw an early-passage stock of your target cells of interest
(hTERT-RPE cells in the example given here). Passage cells
1–2 days prior to infection so that they are ∼30% confluent
in a T-75 flask on the day of infection (see Note 11).
2. Wash the cells twice with 5 ml of HBSS. Add 6 ml of D-
MEM/F:12 supplemented with 10% FBS and 0.1 mg/ml
penicillin-streptomycin (complete D-MEM/F:12) + 6 ml of
your rAAV preparation. Incubate at 37◦C in a tissue culture
incubator for 4 h.
3. Bring the volume up to 15 ml with complete D-MEM/F:12.
Allow infection to continue for 48 h at 37◦C in a tissue
28 Berdougo, Terret, and Jallepalli
4. Plate the rAAV-infected cells at sufficient dilution into
15 × 96 well plates to obtain no more than one clone
per well. To do this, remove the medium from the
flask and trypsinize the cells. Transfer the trypsinized
cell suspension into a tissue culture vessel containing
300 ml of complete D-MEM/F:12+ 0.4 mg/ml G418.
Mix well by gentle inversion or pipetting up and down
and pour the cell suspension into a sterile basin. Dispense
200 ?l/well into 15 × 96-well plates using a repeat pipet-
ter with an 8-port manifold attachment or a multichannel
5. Wrap the stack of 96-well plates in plastic wrap and incubate
at 37◦C in a tissue culture incubator for 2–3 weeks until
colonies form (see Note 12).
3.5. Consolidation of
Colonies and gDNA
1. Two weeks after plating, inspect the 96-well plates for
colonies using an inverted bright field microscope (see
2. To consolidate colonies for PCR screening, carefully aspirate
the media from the plates using an 8-port manifold attached
to a vacuum line. Apply 50 ?l of 0.05% Trypsin-EDTA using
a P200 multichannel pipette and incubate plates for 10 min
at 37◦C in a tissue culture incubator.
3. Using an inverted brightfield microscope, verify that the
colonies are fully detached from the well. Using a multichan-
nel P200 pipette, disaggregate the cells and transfer 40 ?l
from each well to a deep-well block. We regularly pool 5–10
plates per deep-well block in order to reduce the number
of PCR reactions required for the initial screen. However,
single clones can also be analyzed if desired (see Notes 14
4. Using the P200 multichannel pipette or a repeat pipet-
ter with a 50 ml Combitip and 8-port manifold, refeed
the 96-well plates with 190 ?l of D-MEM/F:12 complete
medium per well. Cover the plates in plastic wrap to min-
imize evaporation and return to the 37◦C tissue culture
5. To prepare gDNA, add a sufficient quantity of HBSS to each
well of the deep-well block to bring it to a final volume
of 300 ?l. Purify gDNA using the Wizard SV 96 genomic
DNA purification kit (Promega). It is recommended that the
eluted gDNA be used immediately for PCR screening, but if
this is not feasible, it can be stored at –20◦C (see Notes 16
3.6. PCR Screen
1. All PCR reactions are done in thin-walled 96-well plates.
The reaction conditions per reaction are as follows:
Gene Targeting in Human Cells29
10 × PCR buffer
10 mM dNTPs
primerF (350 ng/?l)
primerR (350 ng/?l)
platinum Taq polymerase
Prepare a master mix (100 reactions per plate) and dispense
10.5 ?l to each well. Add 2 ?l of gDNA to each reaction
using a P10 multichannel pipette and filter tips. Overlay
each well with a drop of mineral oil and cover the plate
with a piece of sealing film (see Note 18).
2. Place the plate in a thermal cycler and cycle as follows:
94◦C × 30s (1 cycle); 94◦C × 15s, 63◦C × 30s, 70◦C ×
2 min (4 cycles); 94◦C × 15s, 60◦C × 30s, 70◦C × 2 min
(4 cycles); 94◦C × 15s, 57◦C × 30s, 70◦C × 2 min (40
cycles; see Note 19).
3. After completion of PCR reactions, remove the sealing film
and add 12.5 ?l of 2 × DNA loading dye to each reaction.
4. Using a P20 multichannel pipette, load 12.5 ?l of each
reaction onto a 0.8% agarose gel containing ethidium bro-
mide cast with 104 wells (multichannel compatible). Run
the gel at 150 volts for 30–45 min and photograph on a
UV light box.
5. Identify the wells giving rise to correct PCR products (see
Fig. 2.3B, white arrowhead).
6. Gel-purify positive PCR products from step 4 above and
digest with the appropriate restriction enzyme (chosen dur-
ing the design stage in Section 3.2) to assess where recom-
bination occurred relative to the exogenously introduced
loxP site (see Fig. 2.3C and Note 20).
7. Go back to your stack of 96-well plates and trypsinize those
wells comprising each PCR-positive pool with 50 ?l of
0.05% Trypsin-EDTA. Transfer each well to a new well
of a 96-well tissue culture plate and add 150 ?l of com-
plete D-MEM/F:12. Return the plate to the 37◦C tis-
sue culture incubator and grow the cells until they reach
8. Prepare gDNA using the SV96 genomic DNA purification
kit and rescreen individual clones by PCR. These clones
are your targeted heterozygotes (i.e., cells with a floxneo/+
genotype; see Note 21).
9. Expand your heterozygote clones to sufficient quantities
and prepare gDNA using the Blood Mini Kit. Use this
30 Berdougo, Terret, and Jallepalli
Fig. 2.3. Generation of PLK1 conditional-knockout cells. (A) Structure of PLK1 alleles generated after gene targeting,
removal of the neomycin-resistance cassette, and deletion of exon 3. In this instance, the first allele of PLK1 was
targeted with a conditional-null vector (left panel), and the second allele was targeted with a constitutive-null vector (right
panel). (B) Example of a positive “hit” from a genomic PCR screen using F4 and neoR primers. The expected product of
2266 bp is marked by a white arrowhead. (C) ApaI restriction digests demonstrate that recombination between the 5’
homology arm and the chromosomal locus took place upstream of the loxP site between exons 2 and 3; in other words, a
favorable crossover occurred. Fragments of 1107 bp and 1159 bp are highlighted by black arrowheads. (D) Verification
of genotypes by Southern blotting. Genomic DNAs were digested with BamHI and SacI and hybridized with a [32P]-labeled
probe (see Fig. 2.2A). Wild-type (4.8 kb), flox (2.1 kb), Δneo (3.2 kb), and Δ (1.6 kb) alleles are indicated with black
arrows. Note that conversion of the PLK1floxallele to a PLK1?allele requires infection with an adenovirus expressing Cre
gDNA for additional PCR and Southern blot analyses to
verify correct recombination at the locus (15).
10. Expand heterozygote clones for long-term cryopreserva-
3.7. Removing the
by FLP Recombinase
and Testing the
Functionality of the
1. Expand heterozygously targeted (floxneo/+) cells to
60–80% confluence in a T-25 flask (see Note 22).
2. Transfect heterozygously targeted cells with the pCAGGS-
FLPe plasmid as follows. In a sterile 1.5 ml tube combine
9 ?l Fugene with 500 ?l of OptiMEM I and incubate at
room temperature for 5 min. Add 3 ?g of pCAGGS-FLPe,
Gene Targeting in Human Cells 31
mix, and incubate at room temperature for 15 min. Wash
the target cells twice with HBSS and replace with 2.5 ml
of complete D-MEM/F:12. Drip the DNA/Fugene mix
onto the cells and return the flask to the 37◦C incubator
(see Note 23).
3. Trypsinize the cells 24 h post-transfection, and plate
into a T-75 containing 3 ?g/ml puromycin. Main-
tain the puromycin selection for 48 h, changing the
medium after 24 h to remove the bulk of dead cells (see
4. After selection, replace the medium with complete
D-MEM/F:12 medium lacking puromycin and allow the
cells to recover at 37◦C in a tissue culture incubator for
several days until they reach about 80% confluence.
5. Trypsinize the cells and prepare dilutions of the cell sus-
pensions so that you can plate the cells using a multichannel
pipette into 2–4 96-well plates at a density of 0.5 cells/well
and 2.5 cells/well.
6. Wrap the plates with plastic wrap and allow them to grow
for 2–3 weeks at 37◦C in a tissue culture incubator until
7. Check the plates for colonies using a brightfield microscope
and identify wells that contain single colonies. Trypsinize
these wells and transfer 48–96 clones to a new 96-well
plate. Add complete medium and expand cells to conflu-
ence. These are your candidate flox/+ cells.
8. Prepare gDNA from the entire 96-well plate as in
9. Set up a PCR screen using a neo-specific primer (neoR;
Fig. 2.2C) and a locus-specific primer to identify clones
that have lost the neo cassette due to FLP-mediated exci-
sion (see Note 25 and Fig. 2.3A).
10. Expand putative flox/+ clones to sufficient quantities
and prepare gDNA using the Blood Mini Kit. Verify
the flox/+ genotype by PCR and Southern blotting (see
11. Prior to the 2nd allele targeting, plate 106flox/+ cells into
complete D-MEM/F:12 medium + 0.4 mg/ml G418 in a
T-75 and allow cells to grow for 2 weeks. Check for com-
plete G418 sensitivity by scoring for any colony growth (see
12. Seed flox/+ cells in a T-25 at a confluency of ∼10%.
The following day, when the cells reach ∼20% con-
fluence, infect with adenoviruses expressing Cre recom-
binase (AdCre (see
13. Remove the AdCre-containing medium 24 h after infection
and replace with complete D-MEM/F:12 medium.
2.4)) at an MOI of
32 Berdougo, Terret, and Jallepalli
14. Harvest the cells 48 h after infection and prepare gDNA
using the Blood Mini Kit for Southern blotting to confirm
the functionality of the loxP site (i.e., deletion of floxed
sequences; see Fig. 2.3A, PLK1?allele).
3.8. Targeting the
1. Thaw and expand early-passage flox/+ cells that have passed
the G418 sensitivity test (described in Section 3.7, step
11) to a T-75 flask, such that they are ∼30% confluent on
the day of rAAV infection.
2. Thaw your rAAV virus preparation made in Section 3.3.
3. Infect flox/+ cells with rAAV particles as in Section 3.4
above (see Notes 27–29).
4. After 2–3 weeks, check for G418-resistant colonies using
an inverted brightfield microscope.
5. Consolidate colonies and prepare gDNA as in Section 3.5.
6. PCR screen colonies and identify individual PCR positive
wells as in Section 3.6.
7. To identify floxed homozygotes (floxneo/flox), perform a
secondary PCR screen on the clones that scored positively
in the first PCR screen, but in this case use locus-specific
primers that span the loxP site, rather than a neo-specific
PCR primer. Gel-purify PCR products and digest with
the restriction enzyme used to mark the loxP site. The
PCR product of a bi-allelic mutant (floxneo/flox) should
cut completely, whereas the PCR product of a monoallelic
(floxneo/+) mutant will cut only partially (less than 50%,
due to random reannealing of the Watson and Crick strands
8. Expand candidate floxneo/flox clones to verify their geno-
type by Southern blotting.
9. Expand candidate floxneo/flox clones for cryopreservation.
10. Generate flox/flox cells by removal of the neomycin cassette
as in Section 3.7.
11. Expand early-passage flox/flox cells to 20–30% confluence
and infect with AdCre.
12. 24 h after infection remove the AdCre containing medium
and replace with complete D-MEM/F:12 medium.
13. Harvest cell pellets at 24 h intervals for several days post
14. Prepare gDNA using the Blood Mini Kit to verify homozy-
gous deletion of the targeted exon by Southern blotting
(see Fig. 2.3D).
Gene Targeting in Human Cells 33
1. Where possible, minimize the repetitive DNA content of
each homology arm to maximize the efficiency of locus-
2. The packaging limit of AAV is 4.7 kb. Targeting vectors
containing sequences larger than this will not package well,
resulting in extremely low titers of transducing virus.
3. The NetPrimer program is invaluable in generating
effective screening primers (http://www.premierbiosoft.
4. The pNY vector is optimized for conditional knockouts, as
marker removal is controlled by the Flp/FRT recombina-
tion system, whereas conditional exon removal is driven by
5. We stress the importance of testing the screening primers in
order to identify those that give a strong and specific PCR
product from very low levels of DNA (typically about 30
copies/reaction). For these tests, each screening primer is
paired with a locus-specific primer that gives a PCR prod-
uct of roughly the same size as that we wish to detect in
the actual screen (2.5–3.5 kb). It may be helpful to vary
the concentration of DMSO while testing your screening
primers to determine optimal amplification conditions.
6. To avoid interference with splicing, we recommend plac-
ing the loxP site at least 100 bp upstream of the 5’ end or
downstream of the 3’ end of the targeted exon. At the same
time, the distance between the loxP site and the neomycin-
resistance cassette should be minimized in order to reduce
the chances of a nonproductive crossover between these
7. The direct juxtaposition of two inverted tandem repeats
(ITRs) results in plasmid instability. For this reason we use
pAAV-lacZ as the source of the pAAV vector backbone.
8. The 5’ and 3’ ITRs in pAAV contain SmaI sites that can
be used to confirm successful transfer of the NotI fragment
from pNY to pAAV. The orientation of the insert does not
seem to affect gene targeting efficiency as both the (+) and
(–) strands are packaged.
9. Transfection of HEK293 cells with AAV producer plasmids
may cause a cytopathic effect, but this is not strictly corre-
lated with high-titer virus production.
10. Freezing the cells should take about 10 min in the dry
ice/methanol bath. Remove the cells from the 37◦C bath
34 Berdougo, Terret, and Jallepalli
when nearly all of the suspension is thawed. In our hands
this takes about 13 min.
11. It is important to use early-passage target cells for rAAV
12. Without removing the plastic wrap, check the stacks 3–5
days after plating to make sure the media is not yellow and
the G418 selection is working.
13. The number of colonies can vary from infection to
infection. We often get anywhere from 300 to 1500
colonies per experiment. Of these, typically 1–10% repre-
sent locus-specific recombinants. We find that HCT116
cells form compact colonies, whereas hTERT-RPE colonies
are looser in structure because of the greater motility of this
14. The number of plates that you pool will depend on the
total number of G418 colonies obtained in the experiment.
If you have ∼1 colony in every well, you will want to pool
a maximum of 5 plates. However, if you have many fewer
colonies, you may want to pool 10 plates. If you consol-
idate more than 5 plates in a deep-well block, be sure to
adjust the volume of cells taken from each well such that
the total volume in each well of the deep-well block is
300 ?l (e.g., for 10 plates, take 30 ?l from each well). Be
sure to label each individual plate with a unique identifier
so that you can accurately deconvolve your PCR positives
from the pool stages.
15. The deep-well block can be stored at –20◦C for a day or
two or used immediately for gDNA preparation. We usually
freeze the cells in the deep-well block for several hours prior
to gDNA extraction to help with the lysis. If storing, cover
the deep-well block with a piece of sealing film. Keep in
mind that your candidate targeted heterozygotes (floxneo/+
cells) will be confluent in several days and so it is critical to
complete the PCR screening to identify positive clones in
this time frame.
16. This protocol is optimized for a centrifuge outfitted with
a swinging-bucket rotor that can accommodate deep-well
blocks. Alternatively, one can use the Vac-Man 96 Vacuum
Manifold with comparable results.
17. After preparing gDNA, we recommend setting up the PCR
screen immediately as the gDNA may not be very stable
and is susceptible to degradation with freeze/thaw cycles.
18. Using mineral oil avoids potential problems with reaction
evaporation and cross-contamination.
19. Extension time may be varied according to the expected
size of the PCR product (1 min per kb).
20. If you are only able to successfully PCR screen across the
homology arm that does not contain the loxP site (i.e., you
Gene Targeting in Human Cells35
cannot determine a favorable crossover), you will have to
expand PCR positive clones and try to rescreen the loxP-
containing homology arm using higher purity DNA pre-
pared by the DNA Blood Mini Kit.
21. You should expect to identify a single PCR positive clone
from each pool you deconvolve. If your initial plates
contain multiple clones per well, a subsequent round of
subcloning by limiting dilution will be necessary to reach
clonality (and confirm heterozygosity) prior to the removal
of the neomycin cassette.
22. We recommend expanding at least two independently
derived floxneo/+ clones for FLP mediated excision of the
neo cassette in order to eliminate clonal bottlenecks in
23. It is helpful to transfect an extra flask of floxneo/+ cells
with a GFP-expressing plasmid, which provides both an
indication of transfection efficiency and a negative control
for puromycin selection. For HCT116 cells we transfect
pCAAGS-FLPe using the Lipofectamine Plus protocol as
described in Section 3.3, as this gives a higher transfection
efficiency than Fugene in this cell type.
24. We find that the puromycin selection is more effective for
HCT116 cells than RPE cells. Because of this, we typi-
cally screen more FLP-transfected RPE clones (96) than
HCT116 clones (48) in order to ensure recovery of neo-
25. It can be helpful to perform a control PCR using locus-
specific primers to confirm that your gDNA is present and
of sufficient quality for screening.
26. Occasionally one may detect the presence of a small frac-
tion of G418-resistant cells in a putatively neo-excised
clone, presumably through low-level contamination by the
parental clone or a neo-positive sibling. In our experience,
such contamination can always be cured by an additional
round of limiting dilution.
27. The amount of virus used in the second round of infec-
tion can be adjusted depending on the number of colonies
obtained from the first round of infection. However, appli-
cation of large amounts of virus (in excess of 6 ml per T-75)
may also increase the risk of multiple integration events.
28. In general, homology arms do not have to be made from
DNA that is isogenic to the target cell line. However, in
rare cases where substantial polymorphisms exist, it is pos-
sible that these sequence differences may result in preferen-
tial and recurrent integration into one allele. In this unusual
situation, we have found that reconstructing targeting vec-
tors based on the specific polymorphisms present in the
second (untargeted) allele can overcome this allele bias and
36 Berdougo, Terret, and Jallepalli
generate a homozygous knockout cell line (M.-E.T. and
P.V.J., unpublished data).
29. It is also possible to target the second allele with a mod-
ified construct in which the targeted exon is deleted out-
right, rather than flanked by loxP sites (see Fig. 2.3A, right
panel). Although this requires some additional cloning
steps, it eliminates the need to consider where crossovers
occur relative to the targeted exon. This can be beneficial
in instances where the distribution of crossovers obtained
with the first-allele targeting construct is found to be biased
towards nonproductive integrants that failed to incorporate
both loxP sites.
The authors thank Catherine Randall for generously providing
the primary data used in Fig. 2.3. Work in the laboratory of P.V.J.
is supported by grants from the National Institutes of Health
(CA 107342) and the American Cancer Society (RSG-08-093-
01-CCG). P.V.J. is a Pew Scholar in the Biomedical Sciences.
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