protocol

Production of knock-in mice in a single generationfrom embryonic stem cells
Hideki Ukai1, Hiroshi Kiyonari2,3 & Hiroki R Ueda1,4
1Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, Osaka, Japan. 2Genetic Engineering Team, RIKEN Center for Life Science Technologies, Hyogo,

Japan. 3Animal Resource Development Unit, RIKEN Center for Life Science Technologies, Hyogo, Japan. 4Department of Systems Pharmacology, The University of Tokyo,
Tokyo, Japan. Correspondence should be addressed to H.R.U. (uedah-tky@umin.ac.jp).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Published online 16 November 2017; doi:10.1038/nprot.2017.110

The system-level identification and analysis of molecular networks in mammals can be accelerated by ‘next-generation’ genetics,
defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic
makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation,by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cellderived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement
for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens thetime required to produce KI ES mice from about a year to ~3 months. Various kinds of KI ES mice can be produced with a minimized
amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report,
we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation
mammalian genetics in organism-level systems biology studies.

INTRODUCTION
To identify and analyze molecular networks and/or cellular
circuits in organisms, gene knockout (KO) or KI technologies
are powerful tools and are often applied in mammalian reverse
genetics studies. However, to produce KO or KI mice using classic
genetics is demanding with respect to time, space, and effort. In
conventional schemes, after target mutations are introduced into
embryonic stem cells (ESCs) by homologous recombination, themutant ESCs are injected into wild type blastocysts to produce
chimeric mice. To produce mice in which all the cells of the bodycarry KO or KI mutations, a requirement for reliable phenotype
analysis, further crossings of the offspring are required. Thesecrosses take several months to years to carry out, as a period of atleast 3 months is required per generation. Therefore, to comprehensively identify and quantitatively analyze the molecular networks and/or cellular circuits in an organism with minimal effort
requires next-generation genetics, i.e., highly efficient production
technologies for obtaining mice that carry KO or KI mutations
throughout all the cells of the body without crossing.
Methods developed to accelerate KO and KI mouse production.
Recent advances in genome-editing techniques have accelerated
KO studies1–7. We recently established an efficient method forthe generation of mice that carry biallelic KO mutations in allcells of the body in one stage8,9. After identifying a molecule ofinterest, it is also important to create KI animals that have modified proteins. Ideally, these KI animals will have a null phenotype that can be rescued with the expression of a mutant formof the deleted gene. This enables the evaluation of the organismlevel effect of modifying the protein. However, in contrast to theimproved KO rates obtained with one-stage production schemes,
zygotic KI remained inefficient, particularly long-fragment insertion by CRISPR-mediated genome editing (initially a KI rate of
only ~20%10). Several studies have sought to improve the KI rate,
for example, by inhibitor treatments11,12, by using Cas9 protein
rather than Cas9 mRNA13–15, and by using two single-stranded

oligodeoxynucleotides (ssODNs) as a bridge between the large
fragment and the genome16. Although one of these studies
showed an increased genome-editing (KI) rate, an up to ~45%
KI efficiency for live-born pups, when injecting a complex of
Cas9 protein/dual crRNA/tracrRNA into pronuclei14, some of theresults were based on a small number of experiments, and thus
the reproducibility of these recent studies must be further evaluated. In addition, usually not all cells of produced KI mice carry
the KI mutation, so further crossing of the offspring is required
to produce mice that carry KI mutations throughout the body. In
summary, further improvements to overcome editing inefficiency(particularly with a large-fragment KI) in the zygote were neededto achieve one-step generation of KI mice.
An attractive alternative method for producing mice that carry
KI mutations throughout the body without crossing is the twostep production of KI mice as almost completely ESC-derived
mice (ES mice). The advantages of using ESCs are the ease of andflexibility for more complex genome editing (e.g., multi-gene KI)
in in vitro culture, the ability to select for sex, the ease of storage,
and the ability to introduce a loss-of-function mutation in anembryonic lethal gene.
ES mouse production within a single generation was first tried
using the tetraploid complementation method17–24. However, thismethod has several potential drawbacks, such as developmental abnormalities caused by substantial contamination by hostcells24,25, and the difficulty of using inbred ESCs21,23,26. Anothermethod for the generation of ES mice is to inject ESCs into, or toallow ESCs to aggregate with 8-cell embryos27–29. All three germlayers (ectoderm, endoderm, and mesoderm) of the adult miceare developed from the inner-cell mass (ICM) of the blastocyststage embryo. After injection or aggregation, the ESCs show
a strong bias toward integration into the ICM of blastocyststage embryos, and the ICM is occupied by ESCs. As the ESCs
are derived from ICM, the cell–cell adhesion properties of the
ESCs might enhance the commitment of ESCs into ICM30,31.
nature protocols | VOL.12 NO.12 | 2017 | 2513

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Development of a system for the efficient parallel
establishment of KI ESCs that are competent for ES mice
production
To improve the throughput of KI ESC production, we focused on
developing the following three methods: (i) a feeder-free culture
method to eliminate the preparation of the mouse-embryonic
fibroblast (MEF) feeder; (ii) a highly efficient and small-scale
KI protocol to enable parallel gene targeting, and (iii) a simple
colony-pickup method to reduce the work involved in handlinga large number of samples in parallel (Fig. 1).
We carefully optimized the feeder-free culture conditions
under which ESCs retain their ability to efficiently contribute toembryos. We found that good chimerism could be obtained using
amine-coated dishes pretreated with 6-bromoindirubin-3′-oxime(BIO)-containing medium (BIO medium; Fig. 2a,b)37,38. Without
the BIO medium pretreatment, ESCs tend to differentiate over
several passages. Although the mechanism underlying this observation is not fully understood, we speculate that the BIO medium
influences the distribution of the three inhibitors in the 3i culture
medium by changing the surface charge of the positively charged
amine-coated dish. This idea was based on our finding that ESCs
could be cultured without impairing quality through several passages in 3i medium on type-B gelatin, which is negatively charged
in a neutral culture medium.
Gene-transfer techniques can be roughly classified into two
types: electroporation and chemical-reagent methods. In this step,
we optimized the chemical-reagent method, as it has several features that make it suitable for a small-scale protocol. In particular, it
is scalable and has higher viability than electroporation. Although
the conditions that result in high gene-transfer efficiency usually
decrease cell viability, we found that the viability could be improvedby treating the cells with BSA after transfection (Fig. 2c,d)37. As
a genome-editing tool introduced by chemical transfection toincrease the KI efficiency, we used the transcription activator-like
effector nuclease (TALEN)5,37,39. The DNA cleavage by TALEN
requires dimerization of its FokI endonuclease catalytic domain40.

Highly efficient establishment of knock-in ES cells
competent for ES mouse generation

Although the developmental mechanism of ES mice remains
unclear, 8-cell injection and aggregation methods can generate ES mice with ~0.1% or less contamination by host-embryo
cells. To minimize such contamination, ESCs are established andmaintained in serum-free three-inhibitor/two-inhibitor (3i/2i) +leukemia inhibitory factor (LIF) medium. ESCs of various mousestrains, even from inbred strains such as C57BL/6 (B6), can beproduced with this method and can stably and efficiently be used
to generate ES mice via 8-cell injection, even after many passages
and gene targeting32. An ES mouse method combining 3i/2i + LIFcultured ESCs with 8-cell injection/aggregation has already beenused to make mice that carry KI mutations throughout the bodywithout crossing33–35, indicating that this ES mouse method is
feasible and robust. The combination of the genetic backgroundof the ESCs and host embryos highly influences the efficiency ofthe somatic and germline contribution of the ESCs36. In the caseof ICR mouse embryos and B6 ES cells, a high production efficiency is achieved for chimeric mice. Moreover, it is also possible
to easily determine the contribution rate of ES cells in chimericmice by using the difference in coat color between ICR mice andB6 mice36. Thus, it is preferable to use a combination of ICR
mouse embryos and B6 ES cells.

Production of ES mousewithout crossing© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocolVector construction(targeting vector,
TALEN-L, TALEN-R)

Steps 1–5
1~4 weeks

ES-cell-friendly andhighly efficient transfection of
TALEN and targeting vector

Steps 6–21
~1 hHigh-quality culture
without feeder cells,
and antibiotic selection

Steps 22–33
12–14 dColony picking
or sorting,and screening

Steps 34–45
1dExpansion cultureandeasy genotyping

Steps 46–68
10–12 dInjection of ESCs into
8-cell-stage embryo
Steps 69–89~1 month
Judgment ofcoat color

Figure 1 | Overview of advanced ES-mouse protocol. Workflow for thehigh-efficiency production of a mouse that carries a knock-in (KI) mutation
throughout the body within a single generation. Our workflow is classified
into two parts: high-efficiency establishment of KI ES cells (ESCs) and the
production of ES mice from KI ESCs without crossing. For the high-efficiency
establishment of KI ESCs, there are four main stages: (i) a high-efficiency KI
protocol using a genome-editing tool introduced by ESC-friendly and highly
efficient transfection (Steps 1–21), (ii) a high-quality-culture protocol
using 3i conditions without the time-consuming preparation of feeder cells(Steps 22–33), (iii) an easy single-colony-isolation protocol using a cell
sorter for high-throughput (parallel) applications (Steps 34–36), and (iv) aneasy genotyping protocol that avoids laborious procedures such as Southernblotting (Steps 37–68). To produce a mouse that carries a KI mutation
throughout the body without crossing, ESCs cultured in 3i medium are
injected into 8-cell-stage embryos (Steps 69–89).

Thus, two TALEN molecules must bind to both the right and left
sides of the target site with the appropriate orientation and spacing.
As a result, the dimer recognizes a target-site sequence that is twice
the length recognized by single TALEN molecules. This molecular property also results in higher specificity and fewer off-target
effects. We tested several homology arm lengths for the targeting
vector and concluded that the optimal length was 4–8 kbp37. We
also confirmed that there was less random insertion with a circular
vector than with a linear one37. Using our optimized TALEN-basedtargeting protocol with a circular vector, the efficiency of correct
genome editing is almost 90%37.

2514 | VOL.12 NO.12 | 2017 | nature protocols© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocolab

Cover with
BIO medium© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

cConcentrated
6(2 × 10 /ml)
ESC
suspension>5 h
37 °C, 5% CO2

Replacethe BIO medium
with
3i culture medium

30 min37 °C, 5% CO2

Seed the ESCs5
at 1 × 10 cells per wellof 6-well plate

Pipette up and down
3 times

DNA–polymer
complex

BSA
15 min37 °C, 5% CO2

Transferto 6-well platewith 3i medium

5 min
37 °C, 5% CO2

96-well plate

d

e

Final
concentrationof BSA (%)

Relative number of
viable cells
24 h after lipofection

0

1

0.5

3.4

1.0

7.3

2.0

7.5

f
Colonies onamine-coated dishgFloating colonies

Colonies ongelatin-coated dishCollagenase
0.1 mg/ml, 5 min
37 °C, 5% CO2

Pick by micropipettehSorted single colonies
Coating
on dishSorting

Percentage of
100% ES-mice

Amine

–

84.2

Gelatin–

83.3

Gelatin+

77.8

Single-colonysorting bycell sorter

Figure 2 | Simple and efficient protocols for establishing KI ESCs under feeder-free conditions. (a) Illustration of the dish pretreatment for feeder-free culture.
To maintain ESC’s pluripotency, it is essential to pretreat the surface of amine-coated dishes (or wells) with BIO medium for more than 5 h at 37 °C in 5%
CO2. Replace the BIO medium with 3i culture medium 30 min before starting the ESC culture, and then incubate the plates at 37 °C in 5% CO2. Seed the
ESCs at 1 × 105 cells per well of an amine-coated 6-well plate, and start the culture at 37 °C in 5% CO2. (b) Photograph of representative ES colonies cultured
under the feeder-free conditions. Undifferentiated ESCs form round colonies under feeder-free conditions. Scale bar, 200 µm. (c) Illustration of the highefficiency-transfection protocol. A concentrated ESC suspension (2 × 106/ml) is mixed with the DNA–polymer complex. ESC viability after transfection is
improved by treating the cells for 5 min with BSA before seeding them into the dish. With this protocol, highly efficient gene transfer is achieved without
exposing the ESCs to anything but 3i-culture medium for long periods. (d) BSA concentration-dependent change in the viability of transfected ESCs. The
transfected ESCs were treated with various concentrations (%) of BSA for 5 min. Relative values are shown with viability defined as 1 when no BSA was added.
(e) Illustration of the conventional procedure for picking colonies. ES colonies are picked up using a micropipette under a microscope, and dispensed one by
one into the wells of a 96-well plate. When using the highly efficient KI protocol, the number of colonies picked per construct may be small. Therefore, when
only a few kinds of KI constructs are used, a conventional manual method can be used. (f) Illustration of the procedure for automated-colony dispensing forhigh-throughput applications. Colonies cultured on gelatin-coated dishes are suspended by collagenase treatment (final concentration = 0.1 mg/ml,
5 min). Floating colonies are dispensed one by one into each well of a 96-well plate using a cell sorter. (g) Photographs of representative floating ES colonies
before and after sorting. Scale bars, 200 µm. (h) Effect of sorting and gelatin coating on the chimerism of ESCs. ES colonies were cultured on amine-coatedor gelatin-coated dishes, and then separated into 96-well plates manually using a cell sorter. Colonies after sorting were dispersed into single cells by trypsin
treatment and used for 8-cell injection. The number of 100% ES-derived chimeras relative to the total number of pups was calculated. All animal experiments
depicted here were approved by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch, and all the animals were cared for and treatedhumanely in accordance with the institutional guidelines for experiments using animals.

Single-colony manipulation, i.e., the picking up of a colony
from a culture dish hinders the parallel handling of multiple KI
ESC lines. Therefore, we examined protocols for floating colonies into the culture medium and manipulating them using acell sorter. First, we found that ESCs can be cultured on type-B
gelatin without impairing their ability to contribute to chimericembryos. We then used collagenase to degrade the type-B gelatinand detach the ESCs, and confirmed that this had little effect on
the adhesion between ESCs. Moreover, unlike trypsin, collagenase
is not inhibited by divalent ions and retains its activity in the 3i
medium that is preferable for ESC culture. Therefore, the additionof collagenase to 3i medium causes the ESC colonies to detachfrom the type-B gelatin and to float into the culture medium
while maintaining their colony form. These floating colonies cannature protocols | VOL.12 NO.12 | 2017 | 2515

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol
be automatically dispensed into the small wells of a 96-well plate
one by one with a cell sorter (Fig. 2f–h). By combining thesethree procedures, we created an efficient protocol for establishing KI ESCs.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Overview of the procedure
In this report, we describe the entire procedure for the efficient
production of mice that carry KI mutations throughout the
body. The workflow of this procedure is presented in Figure 1.
It includes the TALEN-mediated efficient KI of a gene cassette
including a puromycin-resistance gene into the ROSA26 locus
in 3i/2i + LIF-cultured B6-ESCs (Steps 1–68), followed by 8-cell
injection (Steps 69–89).
Construction of the targeting (donor) vector and TALEN (Steps
1–5). Targeting vectors are constructed by conventional molecularbiological methods. When arms of an optimal length (4–8 kb) areused, the targeting efficiency is usually > 90%37. However, whenarms of ~1 kb are used, the targeting efficiency decreases to ~50%37.
The linearization of targeting vectors increases the frequency of random integration. Thus, we recommend using circular targeting vectors37. Our KI protocol was optimized using C-terminal-truncated( + 63) TALENs41,42 that are expressed depending on the CAG
promoter43. Target sequences of TALENs can be designed using
TALEN-NT44. TALEN expression vector construction kits can beobtained from Addgene (https://www.addgene.org/TALEN/). We
follow Addgene’s detailed protocol for constructing the TALENs.
Genome-editing tools ideally are easy to use; they have strong
activity as well as sequence specificity, and they permit flexibility of target-sequence design. The CRISPR/Cas9 system may
be suitable as an alternative genome-editing tool2. However, as
the target-sequence length of CRISPR/Cas9 is about half thatof TALEN45, CRISPR/Cas9 has lower sequence specificity than
TALEN. Moreover, flexibility of its target-sequence selection
is not higher than that of TALEN because of restriction by the
PAM sequence46. In addition, as for the strength of activity, i.e.,
genome-editing efficiency, TALEN is not inferior to CRISPR/Cas9
in genome editing of ESCs, in which antibiotic selection can beused37. Thus, although its specificity and flexibility have been
improved by using Cas9 nickase47,48 or Cas9 derived from different organism species49,50, using TALEN might have more advantages at the present time.
Although we used a targeting vector with long homology arms forhomologous recombination (HR)-mediated KI, using vectors withextremely short microhomology sequences (10–40 bp) for microhomology-mediated end joining (MMEJ)-mediated editing is an
attractive alternative51,52. For example, the TAL-PITCh (precise integration into target chromosome) system53–55 that combines TALEN
and MMEJ-assisted KI was reported. However, as this system’s KI
efficiency is < 67%55, further improvement is necessary.
Establishment of KI ESCs (Steps 6–33). We use a commerciallyavailable 3i medium (iSTEM, Takara Bio) for ESC culture. By
culturing ESCs on amine-coated dishes that have been pretreated with BIO medium37 for more than 5 h at 37 °C in 5% CO2(Fig. 2a), the cells can undergo > 10 passages without any impairment in their ability to contribute to chimeric embryos. Under
these culture conditions, the ESC colonies maintain a clear spherical shape (Fig. 2b).

We use a commercially available transfection reagent (XfectTransfection Reagent, Takara Bio) to transfect the targeting vector and TALEN-expression vectors into ESCs, using a protocol
modified from that provided by the manufacturer. In our protocol, we treat ESCs similar to nonadherent cells. The DNA–Xfectcomplex is mixed with a single-cell suspension of ESCs by pipetting. The mixture is incubated for 15 min at 37 °C in 5% CO2,
and then BSA is added to a final concentration of 2%37 (Fig. 2c).
The addition of BSA with a final concentration of 1% or more
suppresses the transfection-induced decrease in cell viability(Fig. 2d). The transfected ESCs are dispersed on BIO-treatedamine-coated or gelatin-coated dishes as a single-cell suspension, and then they are selected twice by puromycin with a final
concentration of 1.2 ng/µl. Puromycin-resistant ESC colonies
are isolated into the wells of a 96-well plate one by one with amicropipette or a cell sorter (Fig. 2e,f). The culture on gelatinand sorting with a cell sorter do not greatly affect the ESCs’
morphology or their ability to convert to chimeric embryos(Fig. 2g,h). After trypsinization of ESC colonies, an aliquot ofcells is lysed for screening, and the remaining cells are further
cultured for expansion.
Screening for ESCs that underwent homologous recombination (Steps 34–68). Homologous recombination can bedetected by PCR of the right (3′) homology arm region in celllysates, using a pair of primers that anneal outside the homologous arms and within the inserted cassette 37 (P1 and P2 in
Fig. 3a). It is important to carefully determine the PCR conditions for accurate and sensitive detection. For this purpose,
we construct a control vector that contains the complementary sequence for the P2 primer on a longer right homology
arm, which is not on the targeting vector (Fig. 3b). We thenprepare a dilution series of the control vector in a solution
containing the genomic DNA of the host ESCs. Using this dilution series as a template, we determine the PCR conditions
capable of amplifying the desired single band from 0.5 fg ofcontrol vector contained in 10 ng of host ESC genomic DNA(Fig. 3c)56. Under these optimized PCR conditions, we then use
the above cell lysates of puromycin-resistant colonies as a template, and detect the homologously recombined right homology
arm (Fig. 3d). We then expand the positive clones.
After expanding and storing the ESCs, ESC genome samples
are prepared and used for further confirmation of genome
integrity in the targeted region. For this test, genomic PCR is
performed using a series of primers that anneal outside thehomologous arms and within the inserted cassettes (P1 to P4
in Fig. 3a). Successful targeting in a single allele is confirmed
by detecting all the PCR bands from the wild-type (WT)
allele (P3 and P2), targeting the 5′- (P3 and P4) and 3′-homology arm regions (P1 and P2), and measuring the copy numberof the puromycin-resistance gene37 (Fig. 3e). By these steps, it ispossible to quickly and accurately select clones that have recombined, without relying on the conventional Southern hybridization method.
Injection of KI ESCs into 8-cell-stage embryos to produce
KI ES mice (Steps 69–89). This section provides the detailed
procedure for the 8-cell injection used to produce KI ES
mice (Fig. 4).

2516 | VOL.12 NO.12 | 2017 | nature protocols© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocolaNontargeted
alleleTargetedallele

P3

P2

d
M

P1
KI construct

P2

PCR products

1 2 3 4 5 6 7 8 9 1011 1213141516b

P1
Targeting
KI constructvectorc P1eControl vector

P2

Puro 1-copy
control line (C1)Mineral oil
mES medium drop
Injection drop
ES cells drop
Silicone gasket
Glass slide

Puro 2-copy
control line (C2)Gene Gene Gene
Tbp
A
B
C

Puror

ES genome
DNA (10 ng)

cTargeted ES cloneM

PCR products
Template: mixtures ofES genome (10 ng) with
control vector (fg)
10
5
1
0.5
0

2
Relative copy number© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

d

P2
P1
KI construct
P4

P3

b
Control
vectora

Homology sequence toleft and right homology arms of TV

Puror/Tbp

10

C1 C2 1
Control line

2

2
3
4
5
6
Targeted ES clone no.
Endogenous gene B/Tbp

10

C1 C2 1
Control line

2
3
4
5
6
Targeted ES clone no.

Figure 3 | Screening and genotyping procedures for KI ESCs, and typicalresults. (a) Illustration of the primer positions for screening and genotyping.
The thin black lines indicate genomic DNA, and the thick black lines indicate
the sequence corresponding to the homology arms of the targeting vector(TV). Arrows (P1 to P4) indicate the designed positions of the respective
primers. The direction of the arrow indicates 5′ to 3′ direction.
(b) Illustrations of the right homology arms of the targeting vector
and control vector. The control vector contains a longer genomic DNA
homologous sequence than the targeting vector. The P2 primer homologous
sequence is contained only in the control-vector arm. (c) Illustration of themethod for determining optimal screening PCR conditions. A dilution seriesof the control vector is prepared in a solution containing the genomic DNAof the host ESCs. Using this dilution series as a template, the PCR conditions
capable of amplifying the desired single band from 0.5 fg of a control vector
contained in 10 ng of host ESC genomic DNA using the P1 and P2 primers
are determined. (d) Typical results of the PCR screening of targeted ESCs.
PCR reactions using 16 puromycin-resistant ES colony suspensions were
performed with the P1 and P2 primers, and the products were separated
by electrophoresis. Normally, 80% or more of the clones are detected as
positive clones. (e) Verification of the number of inserted puromycin genes.
(Top) Schematic diagram of the genotype of control or a targeted ESC line
containing one or two copies of the puromycin gene. (Bottom) Quantitative
PCR was performed for the puromycin gene, the Tbp gene, or an endogenous
gene (gene B in the diagram). The Puro or gene B value was normalized to
the Tbp value. The results of these quantitative PCR assays are shown at thebottom. C1, control cell line containing one copy of the puromycin gene; C2,
control cell line containing two copies of the puromycin gene; M, molecularsize marker of DNA; Puror, puromycin-resistant gene.

Limitations of KI ES mouse production and proposed future
improvements
To establish a highly efficient method for the identification andanalysis of molecular networks and/or cellular circuits in organisms,

Figure 4 | Injection of KI ESCs into 8-cell-stage embryos to produce KI ES
mice. (a) The micromanipulator into which the injection and holding pipettes
are set. (b) Three cold medium-sized drops are made on the injection plate,which is prepared beforehand. The injection and holding pipettes are properlyaligned within the mES-medium drop. ESCs are collected into the injection
pipette in the ESC drop. The ESCs are then injected into 8-cell-stage embryosin the injection drop. (c) ESCs in the injection pipette. ESCs are collected
into the injection pipette. An injection pipette with a suitable inner diametereases the control of the ESCs. Scale bar, 40 µm. (d) ESCs are injected into 8cell-stage embryos so that they fill the perivitelline space. The injection should
not cause the zona pellucida to expand. Scale bar, 40 µm. All the experimental
procedures and housing conditions were approved by the Institutional Animal
Care and Use Committee of the RIKEN Kobe Branch, and all the animals werecared for and treated humanely in accordance with the institutional guidelines
for experiments using animals.

we needed to improve both the throughput and the speed of the
KI mouse production process. We improved the throughput of
KI ES production by optimizing the transfection method, which
is suitable for parallel procedures because it is simple and canbe performed on a small scale. We also improved the throughput by simplifying the ESC culture using a feeder-free protocol.
However, the throughput of the 8-cell injection procedure is still
not very high in the present protocol, and it should be improvedin the future. In particular, it will be necessary to automate theinjection procedure or to develop a highly parallel cell-aggregation method that does not require exceptional skills or expensive
and specialized equipment.
Regarding speed, by combining an efficient KI protocol for ESCs
with an 8-cell injection protocol for 3i-cultured ESCs, we established a rapid procedure for producing mice that carry KI mutations throughout the body within ~3 months. A further increasein speed might be difficult with a two-stage method using genome
editing of ESCs. A possible solution is editing of the zygotic
genome, which enables one-step production of KI animals. The
generation of transgenic mice, e.g., YACs/BACs/PACs-transgenic
mice57, by injection into the zygote is faster than the two-stage
method, as there is no need for ES cell targeting and culture. One
advantage of the transfer of these large fragments is positionindependent transgene expression, so the use of YACs/BACs/
PACs transgenics has been a fundamental approach to rescue ofnature protocols | VOL.12 NO.12 | 2017 | 2517

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocolmutant phenotypes. However, the KI efficiency is low (5–20%57),
and transgenes are unstable (30–80% of transgenes are lost57).
In addition, random insertion of the transgene can also cause
disruption of other genes, complicating phenotypic analysis.
Usually, not all cells of the bodies of the produced KI micecarry the KI mutation, so further crossings of the offspring areneeded to produce mice that carry KI mutations throughout
the body for reliable phenotype analysis. Recent studies of KI
into zygotes using efficient site-specific endonucleases, e.g., the
CRISPR/Cas9 system, have enabled accurate insertion; however,the KI efficiency (20–45%) has still not been improved sufficiently10–14,16. Thus, although one-stage methods require more
substantial amounts of money, space, and effort to produce KI
mice than do two-stage methods, the production of many typesof KI mice is necessary for comprehensive identification andquantitative analysis of the molecular networks and/or cellular

circuits in an organism. Our two-stage method is more suitable
than the one-step method for a comprehensive analysis,
even if it takes time to target the ESCs using the current versionof the protocol.
Another drawback of our protocol is that the ES mouse production
rate can depend on the ‘quality’ of the cultured ESCs. Thus, genomic
instability should be avoided during culture and passages because
chromosomal aneuploidy can cause embryonic death. Although
maintaining the ESCs in serum-free 3i/2i + LIF medium improved
the efficiency of ES mouse production, better culture methods and
ESC quality control should improve the efficiency further. A varietyof treatments, including ERK/p38 inhibitor58; PKC inhibitor59,60;
ROCK inhibitor61,62; and HDAC inhibitors (e.g., trichostatin A,
sodium butylate, or valproic acid63–65; or vitamin C66) have beenreported to improve the potency of ESCs, and inclusion of these ordifferent reagents might further improve efficiency.

MATERIALS
REAGENTS
Animals• We have used ICR female mice (CLEA Japan, cat. no. Jcl:ICR or Charles River Laboratories, cat. no. Crl:CD1(ICR)) as pseudopregnant recipient miceand 8-cell-stage embryo donors. ICR mice are inexpensive, and in combination with B6-derived ES cells, the production efficiency of chimeric mice isalso high. By combining ICR mouse embryos and B6 ES cells, the percentage
contribution of ES cells can be readily recognized on the basis of simple
coat-color markers. ! CAUTION Animal experiments must be performed inaccordance with governmental and institutional regulations regarding theuse of animals for research purposes. All the experimental procedures andhousing conditions were approved by the Institutional Animal Care and Use
Committee of the RIKEN Kobe Branch, and all the animals used to produce
the results shown in this protocol were cared for and treated humanely inaccordance with the institutional guidelines for experiments using animals.
Vector construction• Gateway system (Thermo Fisher Scientific, cat. no. 11791019)• Golden Gate TALEN and TAL Effector Kit 2.0 (a gift from D. Voytas and
A. Bogdanove, Addgene, kit no. 1000000024)
Cell culture and embryo manipulation• 6-bromoindirubin-3′-oxime (BIO) for cell biology (Wako Pure Chemical
Industries, cat. no. 029-16241) ! CAUTION BIO is light sensitive.
• DMSO, Hybri-Max, sterile-filtered, BioReagent, suitable for hybridoma(Sigma-Aldrich, cat. no. D2650)• KnockOut Serum Replacement (KSR) (Gibco, cat. no. 10828-028)• 3i medium (iSTEM; Takara Bio, cat. no. Y40010) ! CAUTION The attachedinhibitors are light-sensitive.
• BSA (Sigma-Aldrich, cat. no. A8806)• ESGRO leukemia inhibitory factor (LIF; Chemicon, cat. no. ESG1107)
 CRITICAL This solution can be stored at 4 °C. Freeze–thaw cycles will
reduce its potency. Refer to the product vial label for the exact expiry
information, and use it until the expiration date.
• DMEM, powder, high glucose (Gibco, cat. no. 12100-046)• DMEM, high glucose, pyruvate (Gibco, cat. no. 11995-073)• D-PBS (with CaCl2 and MgCl2; Sigma-Aldrich, cat. no. D8662)• FBS (HyClone, cat. no. SH30910.03)• Sodium pyruvate (100 mM; Gibco, cat. no. 11360-070)• MEM non-essential amino acid solution, 100× (Gibco,
cat. no. 11140-050)• 2-Mercaptoethanol (Sigma-Aldrich, cat. no. M-3148)• Sodium hydrogen carbonate (NaHCO3; Nacalai Tesque, cat. no. 31213-15)• Hydrochloric acid (HCl; Nacalai Tesque, cat. no. 18321-05)! CAUTION HCl is toxic. Perform all procedures in a fume hood.
• Sodium chloride (NaCl; Nacalai Tesque, cat. no. 31320-05)• Potassium chloride (KCl; Nacalai Tesque, cat. no. 28514-75)• Disodium hydrogen phosphate 12-water (Na2HPO4·12H2O;
Nacalai Tesque, cat. no. 31723-35)• Potassium dihydrogen phosphate (KH2PO4; Nacalai Tesque,cat. no. 28721-55)• EDTA disodium salt dihydrate (EDTA·Na2; Dojindo,cat. no. 345-01865)• 2.5% (vol/vol) Trypsin (10×), no phenol red (Gibco, cat. no. 15090-046)• Gelatin solution, type B, 2% (wt/vol) in H2O, tissue-culture grade, sterile,
BioReagent, suitable for cell culture (Sigma-Aldrich, cat. no. G1393)• Xfect Transfection Reagent (Takara Bio, cat. no. 631321)• Puromycin dihydrochloride from Streptomyces alboniger, powder,
BioReagent, suitable for cell culture (Sigma-Aldrich, cat. no. P8833)• PureYield Plasmid Miniprep System (Promega, cat. no. A1222)• PureYield Plasmid Midiprep System (Promega, cat. no. A2496)• Collagenase, Type 3, filtered (Worthington Biochemical,
cat. no. CLS-3)• Liquid hydrogen fluoride (Stella Chemifa, cat. no. 021049)! CAUTION Liquid hydrogen fluoride is very toxic. Avoid its inhalation andcontact with the skin and the eyes.
• Beeswax (Nacalai Tesque, cat. no. 03915)• KSOM (ARK Resource, cat. no. KSOM(KSOMaa))• Pregnant mare serum gonadotropin (PMSG; ASKA Pharmaceutical,
cat. no. PMSG)• Human chorionic gonadotropin (hCG; ASKA Pharmaceutical, cat. no. hCG)
• Milli-Q water (MQ; Merck, Integral 15 system)
Genotyping• Proteinase K, recombinant, PCR grade (Sigma-Aldrich, cat. no.
03115836001)• UltraPure DNase/RNase-free dH2O (Thermo Fisher, cat. no. 10977-023)• SYBR Premix Ex Taq GC (TaKaRa, cat. no. no. RR071A)• KOD FX Neo (Toyobo, cat. no. KFX-201)• Agarose S (Nippon Gene, cat. no. 318-01195)• Tris-acetate EDTA buffer (10×; TAE buffer; Nacalai Tesque, cat. no. 35430-61)• nexttec 1-step DNA Isolation Kit for Tissue & Cells (nexttec
Biotechnologie, cat. no. 0635020)
Primers for PCR• Primer P1: 5′-AGTCTAATGAGAACACAGGGCCAC-3′• Primer P2: 5′-GGTGAAATGCTTGACTCCTAGACTT-3′• Primer P3: 5′-GACAGGGTCTCTTACTAGCCTAAAC-3′• Primer P4: 5′-TCCATCAGAAGCTGGTCGATC-3′• Puro-F: 5′-CTCGACATCGGCAAGGTGTG-3′• Puro-R: 5′-GGCCTTCCATCTGTTGCTGC-3′• Tbp-F: 5′-CCCCCTCTGCACTGAAATCA-3′• Tbp-R: 5′-GTAGCAGCACAGAGCAAGCAA-3′
EQUIPMENT
Tool for designing TALEN• TAL effector-nucleotide targeter (TALEN-NT) 2.0(https://tale-nt.cac.cornell.edu/)
Tools for cell culture and embryo manipulation• Nunc cell-culture treated multidishes (4-well plates; Thermo Fisher
Scientific, cat. no. 176740)• PureCoat Amine 6-well plate (Becton Dickinson Biosciences, cat. no. 356721)• PureCoat Amine 24-well plate (Becton Dickinson Biosciences, cat. no. 356723)

2518 | VOL.12 NO.12 | 2017 | nature protocols© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol• 96-well black with clear flat bottom plate (Corning, cat. no. 353948)• 96-well clear round-bottom plate (Corning, cat. no. 353077)• 15-ml conical tube (Corning, cat. no. 352096 or 188271)• 50-ml conical tube (Corning, cat. no. 352070 or 227261)• Bottle-top filter (Nalgene, cat. no. 595-3320)• Millex-GP syringe filter unit, 0.22 µm (EMD Millipore, cat. no.
SLGP033RB)• Multipurpose refrigerated centrifuge (Tomy Seiko, cat. no. LX-130)• Low-speed refrigerated centrifuge (Tomy Seiko, cat. no. AX310)• High-speed refrigerated microcentrifuge (Tomy Seiko, cat. no. MX305)• CO2 multi-gas incubator (Astec, cat. no. SCA-325DRS or Panasonic,
cat. no. MCO-170AICUVH)• Phase-contrast microscope (Olympus, cat. no. CKX41SF or CKX31)• Disposable hemocytometer (Funakoshi, cat. no. 521-10)• pH meter (Horiba, cat. no. F-52)• Electrode (Horiba, cat. no. 9611)• Pre-separation filters (20 µm; Miltenyi Biotec, cat. no. 130-101-812)• Flow cytometer (Furukawa Electric, Perflow Sort model)• Bio-freezing vessel (Nihon Freezer, Bicell model)• Glass capillary (Narishige, cat. no. G-1.2)• Ampule cutter (Maruemu, cat. no. 0510-16)• Microcaps (Drummond Scientific, cat. no. 1-000-0500)• Terumo syringe (SS-20ESZ; Terumo, cat. no. 1-4908-06)• Silicone tube (4 mm × 6 mm; Tigers Polymer, cat. no. SR1554)• 50° Silicone gasket (2-mm thickness × 37-mm outer diameter × 32-mm
inner diameter; Kokugo, custom order)• Glass slide (no. 1; 40 × 50-mm; Matsunami Glass, cat. no. C040501)• Microforge (Glassworks, cat. no. F-1200)• Microgrinder (Narishige, cat. no. EG-401)• Micropipette puller (Sutter Instrument, cat. no. P-1000 IVF)
• Micromanipulation system (Leica, system combining model
nos. DMI6000B, MP-2, and CTR6000)• CellTram vario (Eppendorf, cat. no. 5176000033)• Microscope (Leica, system combining model nos. MZ95
and CLS150XD)
Tools for genotyping• PCR Thermal Cycler Dice Gradient (TaKaRa, cat. no. TP600)• MicroAmp Optical 384-well reaction plate (Thermo Fisher,cat. no. 4309849)• MicroAmp Optical adhesive film (Thermo Fisher, cat. no. 4311971)• QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher, model no.
QuantStudio 7 Flex)• 0.2-ml Hi-8-Dome Cap (TaKaRa, cat. no. NJ301)• 0.2-ml Hi-8-Tube (TaKaRa, cat. no. NJ300)• 1.5-ml microtubes, flat-bottom (Watson, cat. no. 131-415C)• Electrophoresis system (Gel, model no. Mupid-2 Plus)
REAGENT SETUP
Eight-cell-stage embryos For efficient collection of embryos, ICR female mice
are superovulated by injecting them with 5 IU PMSG, and then with 5 IU hCG
48 h later. Afterwards, they are mated with male mice immediately. Collect8-cell-stage embryos from the uteri of 2.5-dpc (days post coitus) pregnant ICR
female mice, and cryopreserve them in liquid nitrogen using the vitrification
method for long-term storage67.

6-bromoindirubin-3′-oxime (BIO) medium To 425 ml of DMEM (highglucose, pyruvate), add 75 ml of KSR, 0.1 mM MEM non-essential aminoacid solution, 0.1 mM filtered 2-mercaptoethanol, 1,000 U/ml LIF, and 45 µlof 2 mM BIO.
 CRITICAL Store BIO medium at 4 °C and use it within 1 month.
BIO solution (2 mM) Dissolve 1 mg of BIO powder in 1,403 µl of DMSO.
 CRITICAL Prepare aliquots of the stock in tightly sealed vials; store themat − 20 °C and use within 1 month.
BSA solution (1% or 10% (wt/vol)) Dissolve 0.1 mg or 1 mg BSA powderin 10 ml of PBS (without CaCl2 or MgCl2), and filter it using a Millex-GP
syringe filter unit. Prepare aliquots of the stock solutions in vials; store themat − 20 °C and use them within 1 year.
LIF solution (×1,000 stock solution) Dilute the LIF (10 million units/1 ml)
in 9 ml of 1% (wt/vol) BSA.  CRITICAL This solution can be stored at 4 °C
and should be used within 1 year. Freeze–thaw cycles will reduce its potency.
Filtered 2-mercaptoethanol solution Add 14 µl of 2-mercaptoethanol
to 20 ml of MQ, and filter the solution using a Millex-GP syringe filter unit.
 CRITICAL Store this solution at 4 °C, and use it within 1 week.
3i Medium After thawing the iSTEM, add the included inhibitors and
1,000 units/ml LIF.  CRITICAL Store 3i medium at 4 °C and use it within
2 weeks.
mES medium Dissolve the DMEM powder in ~900 ml of MQ, and add
3.7 g of NaHCO3. Adjust the pH (7.2–7.4) using HCl, and dilute the mixture
to 1,000 ml with MQ. After filtering the mixture using a bottle-top filter, add
100 ml of FBS, 1 mM sodium pyruvate, 0.1 mM MEM non-essential aminoacid solution, 0.1 mM filtered 2-Mercaptoethanol, and 1,000 units/ml
LIF.  CRITICAL Store the mES medium at 4 °C and use it within 1 month.
PBS (without CaCl2 or MgCl2) Dissolve 8 g of NaCl, 0.2 g of KCl, 2.89
g of Na2HPO4·12H2O, and 0.2 g of KH2PO4 in ~800 ml of MQ. Aftercomplete dissolution, adjust the pH (7.2–7.4) using NaOH, and dilute thesolution to 1,000 ml with MQ. Sterilize the solution by autoclaving before
use. Store the solution at room temperature (23 °C–26 °C) or at 4 °C, anduse it within 1 year.
EDTA–PBS Dissolve 8 g of NaCl, 0.2 g of KCl, 2.89 g of Na2HPO4·12H2O,
0.2 g of KH2PO4, and 2.2 g of EDTA·2Na in ~800 ml of MQ.
After complete dissolution, adjust the pH (7.2–7.4) using NaOH,
and dilute the solution to 1,000 ml with MQ. Sterilize the solution
by autoclaving before use. Store the solution at room temperature or at 4 °C,
and use it within 1 year.
0.25% (vol/vol) Trypsin Dilute 2.5% (vol/vol)trypsin 10 times with
EDTA–PBS. Store the solution at 4 °C for up to a week.
Puromycin solution (2 mg/ml) Dissolve 100 mg of puromycin powderin 50 ml of MQ, and filter the solution using a Millex-GP syringe filter
unit. Prepare aliquots of the solution and store them at − 20 °C for up
to 1 year.
2 mg/ml Collagenase, type 3 Dilute 50 mg of collagenase, type 3 with
D-PBS (with CaCl2 and MgCl2). After incubating the mixture for 1 h atroom temperature, prepare aliquots and store them at − 20 °C for up to
1 year.
Freezing medium (2×) Dilute DMSO to 20% (vol/vol) with 3i medium.
The solution should be freshly prepared and kept on ice until use.

PROCEDURE
Construction and preparation of the targeting (donor) vector and TALEN-expression vectors ● TIMING 1–4 weeks
1| Construction of TALEN-expression vector. Design the target sequence of TALEN using TALEN-NT 2. 0 (ref. 44;
https://tale-nt.cac.cornell.edu/).
 CRITICAL STEP For targeting into the ROSA26 locus, we use TALENs that bind to the target sequence (5′-CTGCAACTCCAGTCTTTCTAGAAGATGGGCGGGAGTCTTCTGGGCAGGCTTA-3′; TALEN-binding sequences are indicated with italics). The target
sequence was designed using TALEN Targeter for C-terminal-truncated ( + 63) TALENs. As the architecture of TALENs
affects the length of the spacer between the TALEN-binding sequences, it is necessary to design the target sequenceaccording to the architecture of the TALEN used.
2| Construct a pair of TALEN expression vectors (TALEN-L and TALEN-R) using construction kits68 available from Addgene(https://www.addgene.org/TALEN/). Construct the vectors according to the detailed protocol provided by Addgene.
nature protocols | VOL.12 NO.12 | 2017 | 2519
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protocol
3| Construction of the targeting (donor) vector. Clone the regions of 4 kb or more in length on both sides of the
TALEN-cleavage site of genomic DNA into arbitrary vectors as the 5′-homology arm and the 3-′homology arms, in thesame direction and in the same order as the genomic sequence37,69.
 CRITICAL STEP Both homology arms must be designed to not be digested by TALEN. The digestion of homology arms can
be avoided by designing the inner termini of the homology arms (3-terminal of the 5′ homology arm and 5′-terminal of the
3′ homology arm) between the recognition sequences of the right and left TALEN.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

4| Clone the gene of interest and drug-resistance gene into a position between the homology arms to complete thetargeting vector construction. If it is necessary to change the insert genes, it is convenient to use a commerciallyavailable Gateway system37,70 (Thermo Fisher Scientific).
 CRITICAL STEP As the direction of cloning of the gene may affect the expression level, adjust the direction according to
the purpose of the experiment.
5| Preparation of vector DNA for transfection. Prepare a highly purified targeting vector and TALEN-expression vectors byusing a commercially available DNA purification kit according to the manufacturer’s instructions.
 CRITICAL STEP For transfection, use highly purified endotoxin-free DNA. We use the PureYield Plasmid Mini/Midiprep
System (Promega) to prepare the DNA.
Transfection of ESCs with the targeting vector and TALEN-expression vectors ● TIMING 5 d
6| Add 1 ml of BIO medium to each well of an amine-coated 6-well plate, and then incubate the plate overnight at
37 °C in 5% CO2 (Fig. 2a).
 CRITICAL STEP Although pretreatment longer than one night should be avoided, as it tends to cause the colonies
to float, it is essential to pretreat the wells’ surface with BIO medium for more than 5 h at 37 °C in 5% CO2.
7| Replace the BIO medium with 2 ml of 3i culture medium 30 min before starting the ESC culture, and then incubate theplates at 37 °C in 5% CO2. Seed the ESCs at 1 × 105 cells per well, and incubate the culture at 37 °C in 5% CO2. Each day,replace the 3i medium of the culture with fresh 3i medium (preheated to 37 °C).
 CRITICAL STEP To avoid exposing ESCs to air during the daily medium-replacement work, exchange only ~90% of theculture medium, rather than all the culture medium.
8| 3 d after starting ES culture, add 1 ml of BIO medium to each well of a new amine-coated 6-well plate, to be used
the next day for seeding the transfected ESCs. Then incubate the plate, which contains only BIO medium, overnight at
37 °C in 5% CO2.
9| Replace the BIO medium (Step 8) with 2 ml of 3i culture medium before initiating transfection. Then incubate the plate
at 37 °C in 5% CO2.
10| Preparation of suspended ESCs for transfection. 4 d after starting ES culture, confirm that the cultured ES colonies have agood spherical morphology (Fig. 2b).
 CRITICAL STEP ES colonies that show a spherical form and have less contact with the dish should be used. ES colonies
showing a flat shape should not be used.
11| Aspirate the medium. Add 300 µl of 0.25% trypsin–EDTA and wait until the cells start to detach. This usuallytakes ~1 min at room temperature.
12| Deactivate the trypsin by adding 700 µl of mES medium containing FBS. Gently pipette the medium up and down several
times to generate a single-cell suspension.
13| Harvest the ESCs into a centrifuge tube containing 6 ml of DMEM without serum. Wash the wells with 1 ml ofmES medium containing FBS, and collect the wash solution into the tube containing the ESCs.
14| Centrifuge the ESC suspension (190 g, 4 min, 22 °C), remove the medium, and resuspend the pellet in a small amount of
3i medium by gently pipetting several times.

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protocol© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

15| Adjust the ESC concentration to 2 × 106 cells/ml, and place the cells on ice until use. We have no experience ofthe effects of placing the cells on ice for more than 30 min. Thirty minutes is sufficient time to prepare the transfection
reagent–DNA complex.
 CRITICAL STEP There is no need to rinse the ESCs with PBS (without CaCl2 or MgCl2) before trypsinization, as the 3i
medium does not include FBS, which inhibits the effect of trypsin. Do not overtrypsinize the cells. In addition, take care to
avoid too much pipetting, which can damage the ESCs.
16| Transfection with Xfect Transfection Reagent. Prepare the DNA solution and polymer solution according to the manufacturer’s instructions as follows: In a microcentrifuge tube, dilute 0.9 µg of a circular targeting vector and 1.2 µg eachof TALEN-L and TALEN-R expression vectors with Xfect Reaction Buffer to a final volume of 25 µl. Mix the solution well byvortexing at high speed. In another microcentrifuge tube, add 1.65 µl of Xfect Polymer to 23.35 µl of reaction buffer. Mix
the solution well by vortexing at high speed.
 CRITICAL STEP For transfection, use a high DNA concentration so that the concentration of the reaction buffer in thereaction solution does not decrease.
17| Add the polymer solution to the DNA solution, and mix well by vortexing at high speed. Incubate the polymer–DNA
mixture for 10 min at room temperature.
18| While incubating the polymer–DNA mixture, dispense 150 µl of the cell suspension into a well of a 96-well plate.
19| After incubating the polymer–DNA mixture, add it to the cell suspension in the well. Immediately after thisaddition, pipette the suspension up and down three times gently to thoroughly mix the polymer–DNA mixture withthe cell suspension (Fig. 2c).
20| Incubate the ESCs for 15 min at 37 °C in 5% CO2, then add 50 µl of 10% BSA, and mix the solution by pipetting.
Incubate the ESCs for 5 min at 37 °C in 5% CO2.
21| Seed all the cell suspension into the wells prepared in Step 9. Incubate the ESCs for 3 d at 37 °C in 5% CO2.
Each day, replace the 3i medium in the culture with fresh 3i medium (preheated to 37 °C).
Antibiotic selection of transfected ESCs ● TIMING 5 d
22| 2 d after transfection. On the day before initiating antibiotic selection of the transfected ESCs by puromycin,
add 1 ml of BIO medium to each new well of the same or a new amine-coated 6-well plate. Then incubate the plate
overnight at 37 °C in 5% CO2.
 CRITICAL STEP When the automation protocol (Step 36B) is used for single-colony isolation after antibiotic selection,
each new well of the same or a new 6-well plate should be pretreated with 1 ml of 1% gelatin (Fig. 2f) instead of
BIO medium (Fig. 2e) in this step.
23| Replace the BIO medium with 1.5 ml of 3i culture medium before initiating the antibiotic-selection step (next step).
Then incubate the plate at 37 °C in 5% CO2.
 CRITICAL STEP If the wells of a plate were pretreated with 1% gelatin instead of BIO medium in Step 22,
replace the gelatin in this step.
24| Antibiotic selection (first). Aspirate the medium from the well containing transfected ESCs (from Step 21). Add 300 µl of
0.25% trypsin–EDTA to the well, and wait until the cells start to detach.
25| Add 700 µl of mES medium. Pipette the mixture up and down several times gently to generate a single-cell suspension.
Filter the ESCs through pre-separation filters (20 µm, Miltenyi Biotec) and harvest them into a centrifuge tube. Wash thewell with 1 ml of mES medium, filter the wash liquid, and add it to the ESC-containing tube.
 CRITICAL STEP Before initiating the antibiotic selection (Step 28), it is important to dissociate the culture to single cells
to reduce the number of false-positive puromycin-resistant cells.
26| Centrifuge the ESC suspension (190 g, 4 min, 22 °C), remove the medium, and resuspend the pellet in 0.5 ml of 3i medium (preheated to 37 °C) by gently pipetting several times.

nature protocols | VOL.12 NO.12 | 2017 | 2521
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protocol
27| Seed the entire ESC suspension into a well containing 1.5 ml of 3i medium, and incubate the cells for 5 h at 37 °C in
5% CO2.
28| Five hours after seeding the ESCs, replace all the culture medium with 2 ml of 3i medium containing puromycin(1.2 µg/ml). Then incubate the cells for 24 h at 37 °C in 5% CO2.
29| After 24 h (day 2) of puromycin treatment, replace all the culture medium with 2 ml of 3i medium without puromycin.
30| Replace the medium with fresh 3i medium (preheated to 37 °C), and continue to incubate the cells for 24 h.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

31| Antibiotic selection (second). Replace the medium with 2 ml of 3i medium containing puromycin (1.2 µg/ml).
Then incubate the cells for 24 h at 37 °C in 5% CO2.
32| After 24 h of puromycin treatment, replace all the culture medium with 2 ml of 3i medium without puromycin.
33| Each day, replace the medium with 2 ml of fresh 3i medium (preheated to 37 °C), until the ESC colonies are large
enough to be picked (~7–8 d after the start of antibiotic selection).
? TROUBLESHOOTING
Single-colony isolation and screening for targeted ESCs ● TIMING 1 d
34| The day before single-colony isolation of the ESC colonies, add 0.5 ml of BIO medium to each well of a newamine-coated 24-well plate, to be used for ESC expansion. Once you know your general targeting efficiency, you
can determine the number of colonies to pick. We pick 12–16 colonies per construct.
35| Replace the BIO medium with 1 ml of 3i culture medium before starting the colony picking. Then incubate the plate at
37 °C in 5% CO2.
36| Dispense single colonies into each well one by one by the conventional protocol (option A, Fig. 2e) or the automation
protocol (option B, Fig. 2f).
(A) Conventional protocol for single-colony isolation(i) Add 50 µl of trypsin per well to a 96-well round-bottom plate.
(ii) Replace the culture medium with 1 ml of PBS lacking CaCl2 and MgCl2.
 CRITICAL STEP The activity of trypsin is inhibited by divalent ions.
(iii) Place the plate under a microscope to select undifferentiated healthy colonies.
(iv) Using a P20 pipette set to 10 µl, carefully pick a colony without contamination from other colonies, andtransfer it into trypsin in the 96-well plate. Continue picking colonies for 3 min, and then add 150 µl of mES
medium to each well.
 CRITICAL STEP Do not use a multichannel pipette to add the mES medium; instead, use a single-channel pipette to
add the medium at the same rate as the colonies had been picked into each well. Repeat Step 36A(iv) if the desired
number of colonies was not picked within 3 min.
(B) Automation protocol for single-colony isolation(i) Add 100 µl of collagenase (2 mg/ml) to the culture medium, and incubate the cells for 5 min at 37 °C in 5% CO2.
 CRITICAL STEP To apply this method, the ESCs are cultured on gelatinized dishes.
(ii) Confirm that the cultured ESC colonies are floating and maintaining a spherical morphology (Fig. 2g).
(iii) Collect the entire culture into a conical tube (50 ml).
(iv) Dilute the culture to an appropriate concentration with PBS (without CaCl2 or MgCl2).
(v) Using a cell sorter, automatically dispense each floating colony with a diameter of ~100 µm into separate wells of a
96-well round-bottom plate, one by one, using the procedure described in the instrument’s instruction manual. We use
a commercially available sorter, Perflow Sort (Furukawa Electric).
(vi) Centrifuge the 96-well plate (190g, 4 min, 22 °C), and then remove the medium using a multichannel pipette.
(vii) Add 50 µl of trypsin to each well using a multichannel pipette, and incubate the plate for 3 min atroom temperature.
(viii) Add 150 µl of mES medium to each well using a multichannel pipette.
37| Pipette the medium up and down several times using a multichannel pipette to break up the colonies.

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protocol
38| To create a sample for screening (Steps 41–45), prepare 100-µl aliquots from each sample in 8-strip PCR tubes using a
multichannel pipette, and place the tubes on ice until use. Then, transfer the remaining ESC suspension (~100 µl) to eachwell of the 24-well plate prepared in Step 35, and continue culturing the cells for future use; e.g., stocking, genotyping, and8-cell injection.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

39| Centrifuge the 8-strip PCR tubes containing the ESCs for screening (2,000g, 10 min, 4 °C), and remove the medium using a multichannel pipette set to 90 µl.
! CAUTION To avoid losing pellets, leave a small amount (~10 µl) of culture medium.
40| Add 100 µl of cold PBS to each tube using a multichannel pipette. Then centrifuge the tubes (2,000g, 10 min, 4 °C),and remove the PBS using a multichannel pipette set to 100 µl.
! CAUTION To avoid losing pellets, leave a small amount (~10 µl) of culture medium.
 PAUSE POINT Unless the screening work is started within a few hours, these pellets should be stored in a freezer ( − 80 °C)
until use. Under this condition, the pellet is stable, but it is better to finish the screening (Steps 41–45) before it is time toexpand the cells (Step 46), which is usually within 3 d.
41| Add 50 µl of proteinase K solution (0.2 mg/ml) to each ESC-containing PCR tube. Then incubate the tube in a thermal
cycler under the following conditions: 55 °C for 30 min, followed by 95 °C for 10 min.
42| Pipette the mixture up and down several times to mix well, and add 5 µl of the mixture to a new 8-strip PCR tube.
43| Identify the targeted ESC clones by PCR and agarose gel electrophoresis according to preoptimized conditions (Fig. 3c).
The conditions for detecting clones targeted at the ROSA26 region are shown below as an example. First, mix the following
components in a PCR tube:
Component

Amount (l)Final concentration

Colony lysate

5

2 × PCR buffer for KOD FX Neo

12.5

1×

2 mM dNTPs

5

0.4 mM

10 µM Primer P1

0.75

0.36 µM

10 µM Primer P20.75

0.36 µM

KOD FX Neo (1 U/µl)

0.4

16 mU/µl

ddH2O

0.6

Total

25

44| Place the reaction tube in a thermal cycler, and run the following program:
Cycle no.

Denature

1

94 °C, 2 min

2–33

98 °C, 10 s

Anneal and extend

68 °C, 3 min 50 s

45| Run the PCR products on a 0.6% (wt/vol in TAE buffer) agarose gel, and confirm the presence of the intended amplicon(Fig. 3d).
? TROUBLESHOOTING
Expansion culture and genotyping of targeted clones ● TIMING 12–14 d
46| Repeat the passaging of cells from Step 38 that were confirmed to have the intended amplicon, until the numberof cells of each clone reaches the appropriate cell number for a 6-well cell culture plate.

nature protocols | VOL.12 NO.12 | 2017 | 2523
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protocol
47| Seed all the cells on a well of a BIO-treated 6-well plate and continue culturing them.
48| On the day before cell freezing (Step 51), add 1 ml of BIO medium to each new well of an amine-coated 6-well plate,
into which the ESCs will be seeded (Step 55) to prepare the genomic DNA (Step 62). Then incubate the plate overnight at
37 °C in 5% CO2. Next, on the day of cell freezing, replace the BIO medium with 2 ml of 3i culture medium before initiating
the ESC harvest. Then incubate the plate at 37 °C in 5% CO2 until use.
49| Confirm that the cultured ESCs have a good morphology and have reached a subconfluent state.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

50| Preparation of freezing medium. Freshly prepare the freezing medium (2×) containing 20% DMSO immediately before use,
and keep it on ice.
51| Freezing of ESCs. Aspirate the medium, then add 300 µl of 0.25% trypsin–EDTA, and keep the sample at room temperature until the cells start to detach.
52| Deactivate the trypsin by adding 700 µl of mES medium containing FBS. Pipette the medium up and down several times
gently to generate a single-cell suspension.
53| Harvest the ESCs into a centrifuge tube. Wash the well with 1 ml of mES medium containing FBS, and collect the wash
fluid into the tube containing the ESCs.
54| Centrifuge the ESC suspension (190g, 4 min, 22 °C), remove the medium, and resuspend the pellet in a small amount of
3i medium by gently pipetting it several times.
55| Adjust the concentration of ESCs to 2 × 106/ml. Seed the ESCs (5 × 105 cells/well) into the BIO-coated wells prepared in
Step 48.
56| Gradually add an equal volume of cold 2× freezing medium to the remaining ESC suspension, and mix the suspension
by pipetting it up and down several times.
57| Quickly prepare 1-ml aliquots of the cell suspension in freezing medium in labeled cryovials, and place them into a
Bicell container (a freezing container provided by Nihon Freezer) precooled to 4 °C. Immediately place the Bicell container
into a –80 °C freezer for 1 d, and then transfer the cryovials to a liquid-nitrogen tank for long-term storage.
 PAUSE POINT The ES cells can be stored until the start of the injection work; cells can be stored indefinitely
in liquid nitrogen.
58| Confirm that the cultured ESCs for preparing genomic DNA have reached a subconfluent state.
59| Preparation of genomic DNA. Aspirate the medium, then add 300 µl of 0.25% trypsin–EDTA, and keep the samples atroom temperature until the cells start to detach.
60| Deactivate the trypsin by adding 700 µl of mES medium containing FBS. Pipette the medium up and down several times
gently to generate a single-cell suspension. Harvest the ESCs into a centrifuge tube. Wash the well with 1 ml of mES medium
containing FBS, and collect the wash fluid into the tube containing the ESCs.
61| Centrifuge the ESC suspension (190g, 4 min, 4 °C), remove the medium, and wash the pellet with 300 µl of PBS. Centrifuge the ESC suspension (190g, 4 min, 4 °C), and remove the PBS.
 PAUSE POINT The ESC pellet can be stored in a − 80 °C freezer for up to 1 year before starting the genome purification (Step 62).
62| Using a DNA isolation kit, purify the genomic DNA from the ESC pellet. We use the nexttec 1-step DNA Isolation Kit for
Tissue & Cells (nexttec Biotechnologie) according to the manufacturer’s instructions. Other commercially available products
such as the Wizard Genomic DNA Purification Kit (Promega) can also be used.
63| Confirmation of successful targeting into the correct locus. Genomic PCR is performed using a series of primers that anneal
outside the homologous recombination arms and within the inserted cassettes (Fig. 3a). The conditions for PCR depend on

2524 | VOL.12 NO.12 | 2017 | nature protocols© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol
the primer sets. Below, we show the conditions for detecting the arms targeting the ROSA26 region as an example. First, mix
the following components in a PCR tube:
Component

Amount (l)Amount (ng)Final concentration

100

4 ng/µl© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Purified genomic DNA
2× PCR buffer for KOD FX Neo

12.5

1×

2 mM dNTPs

5

0.4 mM

10 µM Primer Fwa

0.75

0.36 µM

10 µM Primer Reva

0.75

0.36 µM

KOD FX Neo (1 U/µl)

0.4

16 mU/µl

ddH2O

–b

Total

25

aPrimer sets are as follows: for the left homology arm of the targeted allele—Primers P3 and P4; for the nontargeted allele—Primers P3 and P2. bAdjust the total volume of the reaction mixture to 25 µl.

64| Place the reaction tube into a thermal cycler, and run the following program for the left homology arm of the targeted
allele:Cycle no.

Denature

1

94 °C, 2 min

2–31

98 °C, 10 s

AnnealExtend60 °C, 30 s

68 °C, 4 min

Run the following program for the nontargeted (wild-type) allele:
Cycle no.

Denature

1

94 °C, 2 min

2–31

98 °C, 10 s

AnnealExtend58 °C, 30 s

68 °C, 14 min

65| Finally, run the PCR products on a 0.6% (wt/vol in TAE buffer) agarose gel, and confirm the presence of theintended amplicon.
66| Confirmation of successful targeting with a single copy. Perform a quantitative PCR assay using the extracted ESC
genomic DNA and primers that anneal to the coding sequence of the antibiotic-resistance gene. Below, we show theconditions for detecting the puromycin-resistance gene as an example.
First, mix the following components in a well:
Component

Amount (l)Final concentration

Sample genomic DNAa (0.2 ng/µl)b60.08 ng/µl

SYBR Premix Ex Taq GC

7.5

1×

ddH2O

1.02

0.4 mM

100 µM Primer-Fc

0.09

0.36 µM

100 µM Primer-Rc

0.09

0.36 µM

Reference dye

0.3

2%

Total

15

aAs a control, genomic DNA containing one or two copies of the Puro gene is used. bTo make a standard curve, a dilution series of genomic DNA containing one copy of the Puro gene (2, 0.4, 0.08, 0.016, and

0.0032 ng/µl) is used. cPrimer sets are as follows: for the Puro gene—Primer Puro-F and Puro-R; for Tbp—Primer Tbp-F and Primer Tbp-R.

nature protocols | VOL.12 NO.12 | 2017 | 2525
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol
 CRITICAL STEP Although duplicate reactions are sufficient to create the standard curve, triplicate reactions for eachsample are recommended.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

67| Place the reaction plate into a QuantStudio 7 Flex Real-Time PCR System, and run the following program:
Cycle no.

Denature

Anneal1

95 °C, 60 s

2–46

95 °C, 10 s

60 °C, 50 s

47

95 °C, 15 s

60 °C, 60 s

Extend95 °C, 15 s

68| Normalize the Puro value to the TATA box-binding protein (Tbp) value.
 CRITICAL STEP The Puro value normalized to the Tbp value should be ‘1’ if a single copy of the inserted cassette was
harbored in the genome. Notably, cells that are confirmed to have an insertion with 1 or 2 gene copies can provide a usefulsample for control experiments (Fig. 3e).
? TROUBLESHOOTING
Injection of ESCs into 8-cell-stage embryos ● TIMING 6 d
69| After genotyping is complete, add 1 ml of BIO medium to each of 2 wells of an amine-coated 6-well plate.
Then incubate the plate overnight at 37 °C in 5% CO2.
70| The next day, replace the BIO medium with 2 ml of 3i medium at least 30 min before starting to thaw
the ESCs, and then incubate the plate at 37 °C in 5% CO2.
71| Thawing of ESCs. Remove the vial from the liquid nitrogen, and warm it quickly in a 37 °C water bath.
72| When the ice crystals have almost disappeared, use a pipette to transfer the cell suspension aseptically to a
50- (or 15-)ml tube containing 10 ml of mES medium to dilute the DMSO.
73| Centrifuge the ESC suspension (190g, 4 min, 22 °C), remove the medium, and resuspend the pellet in 0.5 ml of
3i medium by gently pipetting it several times.
74| Seed 0.25 ml of the ESC suspension into each well containing 3i medium (Step 70). Incubate the ESCs for 3 d
at 37 °C in 5% CO2.
75| Each day, replace the 3i medium used for the culture with fresh 3i medium (preheated to 37 °C).
76| Preparation of ESCs (day 3). Replace the 3i medium of subconfluent ESCs with 2 ml of 3i medium(preheated to 37 °C), at least 2 h before injection.
 CRITICAL STEP Prepare at least 1,000 times more subconfluent ESCs than the number of embryos to be injected. The mostimportant point is to prepare ES cells in a subconfluent state, as ESCs in the growing phase tend to contribute more to thechimeric mouse. In addition, if there are few cells, this can cause difficulty in manipulating the ESCs during preparation andinjection, so we recommend that you prepare at least 1,000 times more ESCs than the number of embryos to be injected.
These conditions can be achieved by following this protocol.
77| Aspirate the medium, add 300 µl of 0.25% trypsin–EDTA, and wait until the cells start to detach.
78| Deactivate the trypsin by adding 700 µl of mES medium containing FBS. Pipette the medium up and down several times
gently to generate a single-cell suspension. Harvest the ESCs into a centrifuge tube. Wash the well with 1 ml of mES medium
containing FBS, and collect the wash fluid into the tube containing the ESCs.

2526 | VOL.12 NO.12 | 2017 | nature protocols© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol
79| Centrifuge the ESC suspension (190g, 4 min, 22 °C), remove the medium, and resuspend the pellet with a small amountof 3i medium by gently pipetting it several times.
 CRITICAL STEP It is important to dissociate the ESCs to single cells for ease of injection, although care needs to betaken, as too much pipetting tends to damage the ESCs. Resuspending the cells in an excessive amount of medium may makeit difficult to collect the ESCs for injection.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

80| Place the injection and holding pipettes into the micromanipulator setup (Fig. 4a). Fill both pipettes with mineral oil
by applying pressure with the CellTram vario (Eppendorf). Take extra care to avoid air bubbles.
 CRITICAL STEP Air bubbles in the tubing and pipettes make it difficult to control the handling of embryos and the
movement of ESCs.
81| Prepare an injection plate in advance. Apply a 50° silicone gasket (Kokugo) to a 40 × 50-mm glass slide (Matsunami
Glass) with beeswax (Nacalai Tesque) (Fig. 4b).
82| Place three drops on the injection plate (Fig. 4b). The first drop should contain only ES culture medium, the second is
the injection drop (ES culture medium containing 8-cell-stage embryos), and the third is the ESC drop (ES culture medium
containing ESCs). Cover these drops with mineral oil.
 CRITICAL STEP It is necessary to use precooled medium for all three drops. Otherwise, the ESCs will start aggregating andadhering to the bottom of the injection plate.
83| Move the tips of both pipettes into the mES medium drop, and collect a small amount of mES medium.
 CRITICAL STEP At this time, both pipettes should be properly aligned with each other and should be parallel tothe plate surface.
84| Move the injection pipette into the ESC drop, and collect all the ESCs to inject them in one batch(Supplementary Video 1).
 CRITICAL STEP For easy injection, the ESCs should be collected into the pipette with as little space as possible between
them (Fig. 4c). This can be achieved by using an injection pipette with a suitable inner diameter (17–20 µm). If the innerdiameter is too wide, it is difficult to control the ESCs. If the inner diameter of the injection pipette is smaller than an ESC,
the ESCs will be physically damaged, which will impair their effectiveness for chimera mouse production.
85| Move both the injection and holding pipettes into the injection drop. Hold an 8-cell-stage embryo with the holding
pipette, and insert the injection pipette into the embryo.
 CRITICAL STEP Compaction-stage embryos can also be used for the injection. Although the details for theembryo-preparation methods are omitted here, the use of frozen embryos is extremely convenient for injection.
86| Inject 10–30 ESCs into each embryo to fill the perivitelline space (Fig. 4d; Supplementary Video 2).
 CRITICAL STEP The number of injected ESCs should be adjusted according to the size of the perivitelline space. Avoid
injecting an excessive number of ESCs, which causes the zona pellucida to expand. Ideally, one set of injections should befinished in 30 min to 1 h. The number of embryos used for one set of injections should be decided upon depending on theskill of the operator. In our laboratory, 100 embryos are injected within 30 min by one operator.
87| Culture the injected embryos in KSOM-medium drops covered with mineral oil until the next day.
88| Move the (blastocyst) embryos to a KSOM-medium drop that is not covered with mineral oil before transfer.
 CRITICAL STEP It is necessary to remove the mineral oil from the surface of the glass capillary used for embryo transfer.
Mineral oil in the uterus can disturb embryonic development.
89| Transfer the embryos into the uteri of 2.5-dpc pseudopregnant ICR female mice.
? TROUBLESHOOTING? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.

nature protocols | VOL.12 NO.12 | 2017 | 2527
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Table 1 | Troubleshooting table.
Step

ProblemPossible reason

Solution

33 (transfection)

Low number ofcolonies

Poor-quality DNA used fortransfection
Low concentration of DNA
used for transfection
Insufficient TALEN activity

Prepare new DNA for transfection. For transfection, use highly
purified endotoxin-free DNA. We use the PureYield Plasmid Mini/
Midiprep System (Promega) to prepare the DNA. Concentrate the
DNA by ethanol precipitation, and suspend it in Xfect Reaction
Buffer. At least half of the reaction solution used to prepare DNA–
Xfect complexes must be occupied by Xfect Reaction Buffer
Validate the nuclease activity of TALEN by single-stranded-annealing assays with luciferase8. The TALEN for ROSA26 used in thisreport can be used as a positive control for comparing activities

45 (screening of KI
ESCs)

Low percentage
of correct KI ESCs

Inefficient transfection
Insufficient activity of
TALEN

Refer to comments for Step 33
Refer to comments for Step 33

Insufficient selection by
puromycin

Confirm the puromycin concentration and treatment duration. We
used a puromycin-resistance gene that is expressed in a PGK promoter-dependent manner. If different promoters are used, optimization of the conditions may be necessary. Completely dissociate
ESCs into single cells before starting antibiotic selection

68 (genotyping of
KI ESCs)

High percentage
of random integration

Contamination by linearized targeting vector
Puromycin-selection pressure is too highConfirm the form of the targeting vector by electrophoresis, anduse a circular targeting vector. The linearization of targeting
vectors increases the frequency of random integration. Confirm
the puromycin concentration and treatment duration. We used a
puromycin-resistance gene that is expressed in a PGK promoterdependent manner. If different promoters are used, optimizationof the conditions may be necessary

89 (transfer of theinjected embryos)

Low numbers of
2.5-dpc pseudopregnant mice

Unknown causes

Transfer 8-cell-stage embryos to the oviducts of 0.5-dpc pseudopregnant mice. Note that the number of pups may decrease● TIMING
Steps 1–5, construction and preparation of the targeting (donor) vector and TALEN-expression vectors: 1–4 weeks
Steps 6–21, transfection of ESCs with the targeting vector and TALEN-expression vectors: 5 d
Steps 22–33, antibiotic selection of transfected ESCs: 5 d
Steps 34–45, single-colony isolation and screening for targeted ESCs: 1 d
Steps 46–68, expansion culture and genotyping of targeted clones:12–14 d
Steps 69–89, injection of ESCs into 8-cell-stage embryos: 6 d
ANTICIPATED RESULTS
As a reference for the results of the KI experiment, we have provided typical data for inserting a targeting vector containing
8-kb and 4-kb arms into the ROSA26 region37. Using 0.9 µg of the targeting vector under our transfection conditions, several
hundred puromycin-resistant colonies were obtained, and > 80% of the colonies bore the target gene inserted into thecorrect region (Fig. 3d).
The percentage of total pups that were 100% ES mice obtained from ESCs cultured in 3i medium on a BIO-pretreatedamine-coated dish is usually 80% or more (Fig. 2h)37. This percentage tends to be better than when ESCs are cultured on
feeder cells32. Typically, pups are obtained from > 20–30% of the embryos transferred into the uteri of pseudopregnant
mice32,37; so when 80 embryos are transferred, > 16 100% ES mouse pups are usually obtained. After the KI work, someclones sometimes lose their ES mouse competency. As a reference for the ES mouse competency of ES clones after the KI
procedures, we provide typical data for the ES-mouse competency of Cry1 KI-rescue clones37 in Supplementary Table 1.
After the KI procedures, ~50% (29/59) of the ES clones maintained a pup rate (the number of pups per number of transferred
embryos) of 10% or more. In addition, 100% ES mice could be obtained from ~50% (31/59) of the clones. These results
show that 100% ES mice can be obtained by using 2 or 3 KI ESC clones for each targeting construct in the 8-cell injection.

2528 | VOL.12 NO.12 | 2017 | nature protocols© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

protocol
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Acknowledgments We thank our laboratory members at RIKEN QBiC, RIKEN
CLST, and the University of Tokyo, in particular, S. Morino and M. Muramatsu,
for their help in maintaining the ESCs and preparing materials; J. Garçon and
K. Yamanaka for producing ES mice; M. Ukai for help with the genotyping of ESCs;
and M. Kaneko, M. Shigeta and M. Okugawa for help in preparing the manuscript.
This work was supported by grants from AMED-CREST (H.R.U.), CREST (H.R.U.),
Brain/MINDS (H.R.U.), the Basic Science and Platform Technology Programfor Innovative Biological Medicine (H.R.U.), and the Cell Innovation Program(H.R.U.), KAKENHI Grants-in-Aid from JSPS (Scientific Research S, 25221004,
H.R.U.; Scientific Research on Innovative Areas, 23115006, H.R.U.), and thestrategic programs for R&D of RIKEN (H.R.U.), an intramural Grant-in-Aid fromthe RIKEN QBiC (H.R.U.), and grants from Takeda Science Foundation (H.R.U.).
AUTHOR CONTRIBUTIONS H.R.U., H.U. and H.K. designed the study and wrote
the manuscript. H.U. developed, improved, and performed most of the protocols
related to knock-in ESC establishment. H.K. developed most of the 8-cell
injection protocol. All authors discussed the results and commented on the
manuscript text.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html. Publisher’s note: Springer Nature remains neutral withregard to jurisdictional claims in published maps and institutional affiliations.
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