BDR L A R G E G E N E T A R G E T I N G P R O T O C O L(I) Generation of targeting and control vector
We usually perform positive selection using
the neomycin resistance (neor) gene under the PGK
promoter and negative selection with the diphtheria
toxin A fragment gene under the MC1 promoter(Yagi et al., 1990, 1993a). Control vector (CV) isthe vector used to establish the long-sequence PCR
used to identify homologous recombinant colonies
after drug (G418) selection (Murata et al., 2004).
Usually we generate targeting vectors (TVs)that have a longer 5´ homologous arm and a shorter3´ homologous arm. The identification of homologous recombinant colonies is performed by longsequence PCR over the short arm. Consequently,
the CV is also made with the 3´ arm. When one
generates a TV that has shorter 5´ arm and longer

Wild

W kb

MutantX kb

DNA length detected bySouthern hybridization
Y kb

Wild
MutantE2

E1
Wild allele

3´ arm, homologous recombinant colonies are to be
identified by long-sequence PCR over the shorter5´ arm, and the CV should be made with the 5´arm. Attention should be paid that it is frequently
difficult to establish PCR conditions of femtogram(fg) order sensitivity, which is essential for theidentification of homologous recombinant colonies(see Setting PCR condition), by PCR over endogenous GC rich promoter regions or promoters, suchas that of the PGK-1 gene that directs neor geneexpression.
We recommend to construct the CV first andto test the PCR system in advance of constructing
the TV (see Setting up PCR).

Z kb
2nd Ex.

1st Ex.

E1E2

3rd Ex.

PPATGE1

E2
Targeting Vector

E1
Mutant allele

Long armlacZ pA Pro NeopA

lacZ pA Pro NeopA

lacZ pA Pro NeopA

E1

E2PControl Vector

Pro DT-A pA
Short arm

E2
Plox71

ATG

Translation start siteloxP

PProbe for Southern hybridization

frt

E1/E2 Restriction enzyme site for Southern hybridization
Primer for isolating of homologous recombinantFigure 1. Strategy of homologous recombination for conventional KO

(1) Conventional knock-out and knock-in
The homologous arms of the TV and CV areisolated using the Red/ET recombination cloningsystem (Gene Bridges GmbH) and bacterial artificial chromosome (BAC) clone. TT2 cells are F1
hybrid between female C57BL6 and male CBA(Yagi et al., 1993b). We usually obtain BAC clones
from the C57BL/6 BAC library. When using C57
BL/6 BAC DNA we usually expect homologous
mutation to occur in the C57BL/6 allele of the TT2cells, though it must be confirmed with polymorphic markers to be conclusive. When you want tomake the mutation in the CBA allele, you must obtain the BAC clone from the CBA BAC library.
The Red/ET recombination system assembles thetarget genomic DNA of any size and at any chosen
position in the BAC clone into the Minimal Vector(MV) in the E.coli.
DT-ApA/Neo and DT-ApA/lacZ/Neo are thecassettes used to construct TVs to simply knock out
genes and to knock-in the lacZ gene into each locus, respectively (see LARGE home page,
http://www.cdb.riken.jp/arg/: Murata et al., 2004).
Neo and lacZ/Neo cassettes that have no diphtheria
toxin A fragment gene for negative selection are thecorresponding cassettes to construct the CVs. ThelacZ knock-in is designed so that the lacZ gene isincorporated into the 5´ UTR in front of the translational start site of the target gene. Here the construction of the vectors is illustrated with an exam3’

SHS of 5’end
Restriction enzyme site
5’

ple using the DT-ApA/lacZ/Neo cassette.
Using sequence information provided byyou, we will identify the BAC clone by searching
NCBI Blast search(http://www.ncbi.nlm.nih.gov/BLAST/) or UCSC
genome Bioinformatics(http://genome.ucsc.edu/index.html?org=Mouse) ;the BAC clone will be obtained from BACPAC
resources (http://bacpac.chori.org/).
To obtain the 5´ arm two short homologous
sequences (SHSs) of 50bp are chosen; one at about
8kb 5´ upstream that corresponds to the 5´ end of
the 5´ arm (5´SHS) and the other at the site of mutation that corresponds to the 3´ end of the 5´ arm(3´SHS) (Fig. 2). In the knock-in mutation 3´SHScorresponds to the site between the 5´UTR andtranslational start site of the gene. At the 5´ end of
the 5´SHS a restriction enzyme site for either AscI,
AflII or Sal I is generated (we primarily chooseSalI) and at the 3´end of the 3´SHS a site for AflII,
SalI or NotI (we primarily choose NotI).
The 5´SHS is further combined with the 24 bp sequences at the 5´ end of the MV, and the 3´SHSwith the 22 bp sequences of the MV. The modified
MV is amplified by PCR and electroporated into E.
coli that has the target BAC clone expressing pRed/
ET. In the E. coli the 5´ arm is assembled into theMV, and the 5´ arm is cloned into the DT-ApA/
lacZ/Neo cassette by SalI/NotI digestion (Fig. 2).

(a) PCR amplify

Amplified MV
ori Amp

MV as templateori Amp3’Restriction enzyme site
SHS of 3’end(b) Electroporation

5’Red/ET expression plasmid
BAC clone(c) Subcloning

Sal I

Not I

5’arm clone

Nhe I

Xho I

3’ arm clone(d-1) construction of TV

Nhe I

Xho I

3’ arm clone
for CV(d-2) construction of CV

3’ cloning site
Swa I/Nhe I/Xho I

5’ cloning site
Asc I/Afl II/Sal l/Not I(Targeting Vector)

Figure 2. Cloning of homologous region for conventional KI

5’ cloning site
Asc I/Afl II/Sal l/Not I

3’ cloning site
Swa I/Nhe I/Xho I

The 3´ arm of the TV is obtained similarly,
using the 5´SHS at the site of the mutation withSwaI or NheI sites (we primarily choose NheI) and3´ SHS at about 4kb 3´ downstream with NheI or
XhoI sites (we primarily choose XhoI) and cloned
into the DT-ApA/lacZ/Neo cassette at the NheI andXhoI sites. The 3´ arm of the CV must be longer
than the 3´ arm of the TV (we usually make it about
2kb longer), so that the primer used to identify homologous recombinant ES colonies is set up in the2kb extra region (Fig. 1). The 3´ arm of the CV iscloned similarly into the lacZ/Neo cassette; alternatively it can be obtained by long-sequence PCR as
described previously (Murata et al., 2004).
(2) Conditional KO
For conditional KO, three homologous regions must be cloned separately; the 5´ arm, thefloxed-out body, and the 3´ arm (Fig. 3). The
floxed-out body region is sandwiched by loxP sites
and excised by the Cre protein in the targeted allele.

(II) Setting up PCR
After positive and negative selection with aTV, each surviving colony is composed of about
4000 cells. We identify homologous recombinantcolonies with half of these cells (about 2000); theother half of the cells is passaged into one of 24well dish. 2000 cells have about 10 ng DNA in
which one copy of the vector corresponds to a fewfg.
The PCR should be set up so that the longsequence PCR over more than 4kb works at a sensitivity of a few femtograms between the two primers: one primer in the neor insert and the otherprimer at the 3´ site that is included in the CV, but
not in the TV (Fig. 1). Sometimes the difficulty toachieve fg sensitivity can be overcome by changing
primers and PCR condition. We do not recommend
to make a second PCR with the second inner primers, since this usually generate a high number offalse positives. Sometimes, probably due to the sequence composition, the PCR condition at fg senstitivity cannot be established using the 3´ arm in
the design, and as a result the TV must be redesigned. For this reason, we recommend generating
the CV first and testing the PCR condition in advance of constructing the TV.
PCR setups vary by equipment. For the LA PCR
Kit (TaKaRa) we use the following:
PCR buffer: 10×LA PCR Buffer II (Mg2+ free,TaKaRa)
MgCl2: final 1.5 – 2.5 mM
DNA polymerase: LA Taq (TaKaRa)
Pretreatment: at 96 ℃ for 5 min
PCR: 45 cycles (denaturation, 1 min at 96 ℃; annealing and extension 5 min at 68 ℃)
Post-treatment: at 72 ℃ for 15 min.

First PCR conditions are tested with 0.5-10
fg CV DNA. When the sensitivity is obtained at afew fg order, 10ng ES cell DNAs are added to0.5-10 fg CV DNAs, and the sensitivity is confirmed. The PCR condition must be readjusted if
the fg sensitivity is lost with the ES DNAs.
Subsequently, 1000 wild type ES cells areadded to 0.5 -10 fg CV DNAs and the fg sensitivity
is re-confirmed.
When you construct the vector, you have to
make these tests to establish the PCR condition.
Without this, we cannot begin to isolate homologous recombinants.
Finally, the CV is linearized with an appropriate restriction enzyme and electroporated into
the ES cells (see (III) Isolation of homologous recombinant ES cells <Preparation of DNAs>). After
selection against G418, twenty colonies that randomly integrated the CV are arbitrarily picked up
using a 200 μl Gilson pipette, and placed into trypsin solution, after which PCR is performed using
half of the cells (about 2000 cells) of each colony.
About 2000 trypsin-suspended cells of each colony
are washed free of serum with PBS buffer, lysed inwater at 95 ℃ for 5 min, treated with protease K at55 ℃ for 2 h and heat-treated at 95 ℃ for 10 min.
The extract is subjected to PCR. The PCR condition is considered to have been established whenmore than 80% of colonies are PCR-positive.
Even when you construct the vector, we(LARGE) perform this electroporation upon receiptof the CV, and we will send you about twenty colonies of the cells for you to make the PCR.

WildU kb

Neo

V kb

Mutant

W kb

DNA length detected bySouthern hybridization

WildX kb

NeoY kb
Z kb

Mutant
1st ex.

E1

E2

2nd ex.

3rd ex.

E1

E2
PP

Wild allele

E2

Targeting Vector

E1
Pro Neo pA

Pro DT-A pA
about 4kb

about 8kb
Pro Neo pA

Control Vector

E1E2E2

E1
Pro Neo pA

PNeo allele

Pflipase
E1

E2

E1

E2Flox allele
CreE1
mutant allele

E2

E1

E2PP
loxP

Figure 3. Strategy of homologous recombination for conditional KO#"

SHS of 5’end
Restriction enzyme site!"

frt

Probe for southernhybridization
Primer for isolating of homologous recombinant(a) PCR amplify

Amplified MVori Amp

MV as templateori AmpP

E1/E2 Restriction enzyme site for southernhybridization#"

Restriction enzyme site
SHS of 3’end(b) Electroporation!"

Red/ET expression plasmid
BAC clone(c) Subcloning

Sal I

Not I

5’arm clone

Pme I

Nhe I

Sac IIFlox body clone

Xho I

3’ arm clone(d-1) construction of TV
5’ cloning site
Asc I/Afl II/Sal l/Not I

Flox cloning site
Sac I/Pme I/Sma I/Sac II

MC1-DTA-pA

Nhe I

3’ arm clone
for CV

3’ cloning site
Swa I/Nhe I/Xho I

frt

DT-ApA/loxP/frt-Neo

Figure 4. Cloning of homologous region for conditional KOfrt(d-2) construction of CV
3’ cloning site
Swa I/Nhe I/Xho I

PGK-Neo-3’endloxP

Xho I

PGK-Neo-3’end
frt

frt
frt-Neo(III) Isolation of homologous recombinant ES cells
Once the PCR is set up with the CV as described above, it is the time to start the isolation ofhomologous recombinants with the TV. The TV islinearized with the appropriate restriction enzyme
digestion and electroporated into TT2 ES cells(Yagi et al., 1993b).

<Preparation of DNAs>
TV and CV DNAs are purified by equilibrium centrifugation with CsCl-ethidium bromide
gradient; we do not recommend purification using QIAGEN columns. Purified DNAs are linearized with an appropriate restriction enzyme sothat DTA-pA locates at the 3´ end of TV or plasmid sequences at the 3´ end of CV. The completeness of the linearization must be checked bygel electrophoresis. The linearized DNAs arepurified by sequentially treating with phenol
once, with phenol/chloroform twice, and withchloroform once. After the last chloroform treatment, the supernatant fluid must be maintained
under sterile conditions. DNAs are precipitatedwith ethanol and twice rinsed with 70% ethanol.

The DNA precipitates are then dried moderately
and dissolved in HBS buffer at about 60nM concentration. For 10kb DNA, 45μg in 100μl corresponds to about 60nM. Prepare more than 500μl
of 60 nM TV DNA and 300μl of 60 nM CV
DNA and send them to us either at ice-cold temperature or frozen. Indicate your name and thename of the vector on the label.
HBS buffer:
25 mM HEPES
137 mM NaCl
5 mM KCl
0.7 mM Na2HPO4･12H2O
6 mM Dextrose
Adjust to PH 7.0 and sterilize by filtration.

-to be continued<Electoroporation ><Selection and harvest of surviving colonies><Identification of homologous recombinants by long PCR>
<Propagation of potential homologous recombinant clones>(IV) Generation of mutant mice
<Generation of chimeric mice><Generation of heterozygous mutants and breeding of mutant mice for homozygous phenotype analysis>

References
Murata, T., Furushima, K., Hirano, M., Kiyonari,H., Nakamura, M., Suda, Y., and Aizawa, S. (2004).
ang is a novel gene expressed in early neuroectoderm, but its null mutant exhibits no obvious phenotype. Gene Expr. Patterns 5, 171-178.

Yagi, T., Nada, S., Watanabe, N., Tamemoto, H.,
Kohmura, N., Ikawa, Y., and Aizawa, S. (1993a). A
novel negative selection for homologous recombinants using diphtheria toxin A fragment gene. Anal.
Biochem. 214, 77-86.

Yagi, T., Ikawa, Y., Yoshida, K., Shigetani, Y.,
Takeda, N., Mabuchi, I., Yamamoto, T., and , Aizawa, S. (1990). Homologous recombination at cfyn locus of mouse embryonic stem cells with diphtheria toxin A fragment gene in negative selection.
Proc. Natl Acad. Sci. USA 87, 9918–9922.

Yagi, T., Tokunaga, T., Furuta, Y., Nada, S.,
Yoshida, M., Tsukada, T., Saga, Y., Takeda, N.,
Ikawa, Y., and Aizawa, S. (1993b) A novel ES cellline, TT2, with high germline-differentiating potency. Anal. Biochem. 214, 70-76.