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Optimized axolotl (Ambystoma mexicanum) husbandry, breeding,

metamorphosis, transgenesis and tamoxifen-mediated recombination

Article in Nature Protocol · March 2014

DOI: 10.1038/nprot.2014.040 · Source: PubMed

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 protocol

Optimized axolotl (Ambystoma mexicanum)

husbandry, breeding, metamorphosis, transgenesis
and tamoxifen-mediated recombination
Shahryar Khattak1,2, Prayag Murawala2, Heino Andreas1, Verena Kappert1, Maritta Schuez1,2,
Tatiana Sandoval-Guzmán1,2, Karen Crawford2,3 & Elly M Tanaka1,2
1Max Planck Institute ofMolecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany. 2Technische Universität Dresden, Deutsche Forschungsgemeinschaft
(DFG) Center for Regenerative Therapies, Dresden, Germany. 3St. Mary’s College of Maryland, St. Mary’s City, Maryland, USA. Correspondence should be addressed to
E.M.T. (elly.tanaka@crt-dresden.de).

Published online 6 February 2014; doi:10.1038/nprot.2014.040

The axolotl (Mexican salamander, Ambystoma mexicanum) has become a very useful model organism for studying limb and spinal
cord regeneration because of its high regenerative capacity. Here we present a protocol for successfully mating and breeding axolotls
in the laboratory throughout the year, for metamorphosing axolotls by a single i.p. injection and for axolotl transgenesis using
I-SceI meganuclease and the mini Tol2 transposon system. Tol2-mediated transgenesis provides different features and advantages
© 2014 Nature America, Inc. All rights reserved.

compared with I-SceI-mediated transgenesis, and it can result in more than 30% of animals expressing the transgene throughout
their bodies so that they can be directly used for experimentation. By using Tol2-mediated transgenesis, experiments can be
performed within weeks (e.g., 5–6 weeks for obtaining 2–3-cm-long larvae) without the need to establish germline transgenic lines
(which take 12–18 months). In addition, we describe here tamoxifen-induced Cre-mediated recombination in transgenic axolotls.

INTRODUCTION
The application of molecular genetics to the study of classical
regeneration model organisms imparts valuable and detailed
insight into the process of regeneration on the molecular and cel-
lular levels. Axolotls (Ambystoma mexicanum) are an easily bred
species of urodele amphibians. Their high regenerative capacity
combined with the availability of germline transgenesis tools have
established them as the premier organisms for molecular studies
of limb and spinal cord regeneration1–4. One axolotl female will
typically lay 500 eggs in the course of several hours, and through
the injection of several different plasmids into the eggs, multiple
DNA plasmid constructs can be screened for transgenesis using
eggs from a single spawn. A widely used transgenic technique
for Xenopus, fish and salamander species is I-SceI meganuclease–
mediated transgenesis5–8. Successful generation of transgenic
newts was also reported by using intracytoplasmic sperm nucleus
injections mixed with plasmid DNA into unfertilized eggs9.
Salamander researchers have mostly used I-SceI meganuclease–
mediated transgenesis for obtaining transgenic newts and
axolotls5,8,10,11.
DNA transposons have been successfully adapted for trans-
genesis in various fish and mammalian systems8,12–14. We have
established germline transgenic axolotls by using both the I-SceI
meganuclease and the Tol2 transposase5,11,15. Tol2 is an autono-
mous transposase belonging to the hAT family of transposons,
initially identified from the freshwater teleost fish medaka
(Oryzias latipes)16. We have successfully adopted the mini Tol2
system to generate transgenic axolotls. It consists of the minimal
cis-sequence of the Tol2 element that is essential for transposi-
tion17. To successfully establish germline transgenic axolotl lines,
it is necessary to grow and mate founder animals (axolotls) that
express the transgene in the majority (preferably all) of cells of
their body. The mini Tol2 system is more efficient than the I-SceI
method in generating transgenic axolotls that strongly express
transgenes in the majority of cells of founder animals. In the
I-SceI system for transgenesis, the minority founder animals are
grown to sexual maturity to check for germline transmission,
requiring 12–18 months to establish a transgenic line and to
perform experiments. In contrast, Tol2-mediated transgenesis is
efficient and results in a large number of transgenic larvae that
express the transgene all over the body. These animals can be
grown to the desired size and allow experiments to be performed
with F0 (founder) animals within weeks, without the need to wait
for germline transmission.
The limitation of the Tol2 method is that, as the Tol2
transposase is very efficient, it sometimes results in nonuniform
transgene expression (owing to multiple integrations of the plas-
mid DNA) between different cells within the same animal. We
strongly recommend using a fluorescent reporter and prescreen-
ing (on the basis of fluorescence) the founder transgenic larvae for
uniform transgene expression before the start of the experiment
or before raising (growing) them for germline transmission. If
nonuniform expression is observed in all the founder animals,
titration of the transgene DNA and of the transposase RNA will
be necessary to achieve uniform expression of the transgene.
Molecular analysis of regeneration depends on the tissue- and
time-dependent control of gene expression11,18. We have recently
described a set of germline transgenic axolotls for controlling
gene expression in various regeneration-relevant tissues and in a
time-dependent manner by using Cre recombinase11.
We report here a detailed protocol for axolotl husbandry,
breeding and mating, as well as for generating transgenic axolotls
via both the I-SceI and the mini Tol2 systems; we also provide a
quick and easy protocol to metamorphose the axolotls (described
in Box 1).
We have established germline transgenic axolotl loxP reporter
and Cre driver lines for spatial and temporal control of gene
expression11 and successfully crossed and induced exogenous gene
expression via tamoxifen-induced Cre-mediated recombination

nature protocols | VOL.9 NO.3 | 2014 | 529

 protocol

Box 1 | Metamorphosing axolotls ● TIMING 60 min per 20 animals

1. Wet a paper towel (or Kimwipe) in 0.03% (wt/vol) benzocaine and place it in a 145-mm Petri dish.
2. Anesthetize the animals with 0.03% (wt/vol) benzocaine. Fish out the axolotls (>4 cm in length from snout to tail tip) with a
net and gently place them in the 0.03% (wt/vol) benzocaine.
3. Weigh each animal to determine its weight and return it to the wet (benzocaine-soaked) paper in a Petri dish.
4. Measure the desired amount of thyroxine according to 1.5 µl per gram body weight of working stock (1 µg/µl) solution.
5. Prepare the Hamilton syringe with a 30 G, 1/2-inch, 0.3 × 13 mm needle with the desired volume of thyroxine.
6. Inject the thyroxine i.p. by entering the lower abdominal cavity and gently guiding the needle tip into the upper thoracic region of
the animal before injection (Supplementary Fig. 2g,h).
7. Cover the animal with a tissue paper wet with 0.01% (wt/vol) benzocaine and close the lid of the Petri dish to make a
hydration chamber.
8. Keep the animals in the hydration chamber for 30–45 min and then return them to fresh water.
9. Gills and dorsal fin will start to regress 7–10 d after injection. As this occurs, lower the water level such that the animal’s body is
just covered in water.
10. When the gills are completely gone, keep the level of water just low enough to cover the animal. Put an inclined rock at one
end of the tub so that animals can easily climb on it (Supplementary Fig. 1d).
? TROUBLESHOOTING
© 2014 Nature America, Inc. All rights reserved.

in the F1 and F2 generations11. We discuss here how to induce Cre-
mediated recombination by the administration of tamoxifen.
Experimental design
Axolotl husbandry. Axolotls are kept at 18–20 °C in water with
a 12-h light/12-h dark cycle. The temperature is kept constant
within a range of ±0.5 °C with inline cooling systems, as fluc-
tuations in temperature can lead to failure in mating. Changes
in water temperature are monitored daily. For our large colony,
we use continuous-flow aquaria systems in which UV-treated,
biofiltered water is recirculated through the system with 5–10%
of the water refreshed daily. The towers and aquariums should
be equipped with a fine filter (for waste removal), UV light
(against microorganisms) and a biofilter to convert NH4 to NO2
and later to NO3. It is recommended that the level of NO2 be 0
and that of NO3 should be kept below 100 mg per liter. We per-
form an extra change of water and clean the biofilter if the level
of NO3 is >100 mg per liter. Every week, the water is checked
for pH, conductivity, NO3 and NO2 levels. The ideal pH should
be between 7.0 and 8.0. If the observed pH deviates from this,
perform an extra water change every day, or, if the animals are
housed in an automatic water-flow system or aquarium, clean
the biofilter (Supplementary Fig. 1, Supplementary Video 1).
The conductivity of water should ideally be between 500 and
750 microSiemens (µS)/cm.
The water flow into the individual cages should be kept at
restricted levels to reduce irritation to the axolotls’ skin. In our
colony, we keep axolotls that are ≥4 cm in length (from snout to
tail tip) in the continuous-flow aquaria system. The larvae, from
hatching until they reach 4 cm in length, are kept in small plastic
containers as group holdings or in individual cages. The water in
these tubs and individual cages is manually changed three times
a week. We suggest not putting smaller than 4 cm in the auto-
matic water-flow systems to avoid injury to the small animals
from water current or flow.
When setting up a new axolotl colony, it is necessary to check
the ammonia level daily, as the bacteria in the biological filters are
not fully adapted to the system, and in combination with high pH,
they can be harmful to the animals’ health. The boxes and cages
should be cleaned routinely to avoid algae growth. Clean the boxes
with water, brushes, a clean kitchen cloth and/or a sponge; do not
use any soap or chemical detergents during cleaning.
For small-scale colonies, manual changing of water three times
per week, 2 h after feeding, is sufficient. Water should be dechlo-
rinated by overnight evaporation, chemical conditioning or car-
bon filtering. The temperature of water and the room (where
animals are kept) should ideally be between 18 and 20 °C. The
larvae for experiments can be kept at room temperature (up to
24 °C) for the duration of the experiments, but care should be
taken that the room temperature remains below 24 °C for these
larvae, especially during summer months. Never keep the adults
and breeding animals at a temperature higher than 18–20 °C.
The axolotl larvae (from hatching until they reach 5 cm in size,
measured from snout to tail tip) are fed Artemia (brine shrimp),
whereas juveniles (6–20 cm in size) are fed Artemia and small
fish pellets (3 mm) every day. As juveniles grow in size, reduce
the amount of Artemia to completely adapt the feeding regime
to fish pellets only. Adults (21 cm and longer) are fed three times
a week with bigger fish pellets (5 mm). The leftover fish pellets
are removed a few hours after feeding. When feeding Artemia in
the continuous-flow aquaria system, stop the water flow for 2–3 h
to avoid flushing Artemia from the cages (Supplementary Fig. 1,
Supplementary Videos 1 and 2).
Axolotl mating and egg collection. In our experience, a female is
ready to be mated between 12 and 15 months of age. The female
should have a round belly (indicative of having eggs) and should
not be skinny. The females are kept in group holdings. The ear-
liest time point that a male can be mated is ~9–12 months of
age. A mature male can be mated every 4 weeks and the females
can be mated every 8 weeks. Males must be muscular, healthy
and must have a nicely shaped cloaca (Supplementary Fig. 2).
Males are housed separately from each other. Housing them in
a group holding is also possible but may result in a decrease in
mating efficiency. It is also possible to set up mating directly from
group holdings of both males and females without the necessity

530 | VOL.9 NO.3 | 2014 | nature protocols


Figure 1 | Various components of the axolotl transgenics protocol.
(a,b) The plastic mold (a) custom-made at the MPI-CBG machine workshop
is used for preparing agar channels for injecting axolotl eggs as shown in b.
(c) The female axolotl lays eggs on plastic leaves. (d) Spermatophores in a
Petri dish. The opaque white structure contains the sperm. The transparent
jelly supports the attachment of these pockets of sperm to rocks in natural
habitats, or to plastic or glass surfaces in a laboratory setting. (e) The
eggs are manually collected with a scalpel (Supplementary Video 4).
(f) Schematic of the plasmid containing a multiple cloning site (MCS) in
which a transgene cassette of interest is cloned. Note that there are I-SceI
meganuclease sites as well as mini Tol2 sites surrounding the MCS to allow
flexibility of use and provide a possibility to directly compare both the I-SceI
and mini Tol2 system with the same expression cassette. Amp res, ampicillin
resistance; ori, origin of replication. (g) Freshly hatched transgenic larvae.
These are ready to be screened on the basis of fluorescent intensity. Scale
bar, 0.5 cm. (h) Axolotls that have been injected i.p. with 4-OH tamoxifen
for Cre-mediated recombination. Fast Green FCF dye is added to the 4-OH
tamoxifen just before the injection and the resulting green color in the
bellies is indicative of a successful injection. Scale bar, 1 cm.
of keeping them in isolation for 2 d before mating (see mating
© 2014 Nature America, Inc. All rights reserved.
procedure, Steps 1–14). In that case, the mating tanks are kept at
a lower temperature (15–16 °C) than the regular group holding
tanks (18–20 °C). For mating, put the male and female into the
mating tank along with the plastic leaves and shut off the fresh
water flow. We think that the sudden shift in water temperature
and the feeling of fresh water stimulate the animals to mate.
Further information about husbandry and mating can also
be obtained from the following web resources: http://www.
ambystoma.org/education/guide-to-axolotl-husbandry and
http://www.axolotl.org/index.htm.
Metamorphosis of axolotls. Axolotl metamorphosis is described
in Box 1. Axolotls are neotenic and reach adulthood without
undergoing full metamorphosis. If an experiment requires met-
amorphosis of axolotls, the axolotls can be metamorphosed by
the administration of thyroxine to the animals in their rearing
water19 or by a single i.p. injection20. The water-based protocol
is a lengthy process and the chances of cross-contamination of
water with thyroxine are high. In contrast, the single i.p. injection
results in metamorphosed animals within 10–12 d. By using the
i.p. injections in our laboratory, we have successfully metamor-
phosed animals from 4–12 cm in length (from snout to tail tip).
This is usually the size at which most of the regeneration-related
experiments are performed.
Addition of Fast Green FCF to the thyroxine solution reduces
the metamorphosing efficiency. Do not add Fast Green FCF to the
thyroxine solution to be injected. Use a separate net for chang-
ing water and keep the boxes and nets that come in contact with
these animals separate. Animals should be kept under optimal
conditions and checked at least twice a day, as the metamorphic
transition can be fatally stressful for them, and animals can sud-
denly develop blisters on their abdomens. Once the process of gill
and dorsal fin reduction starts, metamorphosis occurs rapidly
and the eyes bulge out from the head surface. Animals may not
eat well during metamorphosis, but one should maintain a daily
feeding and regular cleaning schedule throughout this time. Once
a metamorphosis is complete, the animals will start eating and
digesting food. The animals will shed their skin quite often and
need to be cleaned more than their unmetamorphosed counter-
parts (Supplementary Video 3).
protocol
a b
m
5c
0.5 mm
cm
2.5 mm 3 mm
6.5
2 mm
4 cm
c d
e f F1 ori
Amp res M13 universal primer
Tol2 seq
pBSII-SK-mTol2-MCS
(3,459 bp) Scel site
MCS
Scel site
Tol2 seq
M13 reverse primer
ColE1 origin
g h
Transgenesis of axolotls and tamoxifen treatment for Cre-
mediated recombination. Axolotl transgenesis is achieved by
collecting the freshly laid, one cell-stage fertilized eggs and inject-
ing them with DNA or DNA-RNA mix. A custom-made plastic
mold (Fig. 1a) is placed upside down in a 100-mm Petri dish
containing 10 ml of 2% (wt/vol) agar in water. After the agar
has solidified, the plastic mold is removed to obtain the agar
‘injection plate’. The injection plate has channels to hold the
eggs in place to streamline the injection process, and the agar
bed provides cushioning to the eggs during injections of DNA or
DNA-RNA mix. Freshly collected, dejellied eggs are placed in the
channels and injected with a transgene DNA/I-SceI enzyme mix
or a transgene DNA/Tol2 mRNA mix (Fig. 1b, Supplementary
Videos 4 and 5). The DNA constructs used for making transgenics
must contain a fluorescence reporter to monitor the transgenesis
efficiency. We screen the hatched larvae (from the putative trans-
genic eggs) by virtue of fluorescence expression. We characterize
our transgenic animals as ‘strong’, ‘medium’ and ‘weak’ on the
basis of their fluorescence expression and intensity profile. When
using a ubiquitous promoter (e.g., CMV, CAGGs and others),
a strong phenotype refers to the hatched transgenic larvae in
which the fluorescence gene is expressed in 95–100% of the body.
The medium category refers to fluorescence gene expression
in 50–80% of the cells, whereas weak larvae have <50% of their
body cells expressing the transgene.
The larvae from the injected eggs are screened under the fluo-
rescence microscope for transgene expression. For screening, we
anesthetize the freshly hatched larvae in 0.01% (wt/vol) benzo-
caine. As soon as the animals are anesthetized, we further dilute
nature protocols | VOL.9 NO.3 | 2014 | 531
 protocol
the 0.01% anesthetic (in which the anesthetized animals are) to
0.005–0.0025% with water. Pick up a single animal with the help
of a wide-bore, sterile plastic transfer pipette and gently place it
in a Petri dish with anesthetic. Check it under the fluorescence
microscope and place it back in the fresh water as soon as pos-
sible. The freshly hatched larvae are very sensitive to benzocaine.
We do not anesthetize all the animals at once for screening. We
recommend anesthetizing 2–4 animals, screening them and then
anesthetizing the next batch of 2–4 animals. It is important to
use water that has been dechlorinated by overnight evaporation,
chemical conditioning and/or carbon filtering.
After visual fluorescence selection, the tails and limbs of the
strong animals are cut, fixed, cryosectioned (cross and longitu-
dinal) and then stained with a DNA dye (e.g., DAPI, Hoechst)
to confirm the colocalization of the fluorescence reporter with
stained nuclei and confirm the expression at the cellular level. In
the case of a transgenic axolotl with a tissue-specific promoter
(e.g., muscle-specific promoter), the strong category would refer
to animals in which 95–100% of all the animal’s muscles fibers
© 2014 Nature America, Inc. All rights reserved.
express fluorescent protein (e.g., GFP). For screening the tissue-
specific expression on a cellular or tissue-specific level, the limbs
and tails of the strong animals are cut, fixed and cryosectioned.
The sections are immunostained with respective antibodies
(in this example, muscle-specific antibodies such as myosin heavy
chain (MHC)) to confirm the colocalization of GFP with MHC.
Once a strong founder is confirmed on the cellular level, it is
grown to sexual maturity and checked for germline transmission
by mating it with a nontransgenic animal.
When injecting tissue-specific constructs by using I-SceI–
mediated transgenesis, we do see some ectopic ‘nonspecific’
expression in tissues other than the intended, in F0 animals, for
some but not all of the constructs. For example, we observed
noticeable ectopic expression with a muscle-specific cardiac-
actin:GFP construct, which upon germline transmission showed
muscle-specific expression. Our interpretation of this phenom-
enon is that it is primarily due to expression from nonintegrated,
episomal copies of the injected DNA. Unfortunately, we have not
quantified the percentage of constructs that show this property;
perhaps up to 50% of constructs may show some sporadic expres-
sion in some muscle fibers or skin. Ectopic expression can be seen
with some constructs in the F0 generation, and therefore it is
MATERIALS
REAGENTS
• Axolotls: the loxP reporter and Cre driver lines are available on request from
authors; nontransgenic strains can be purchased from the Ambystoma
Genetic Stock Center (http://www.ambystoma.org/genetic-stock-center)
! CAUTION Appropriate license and approval from the respective ethical
boards (institutional and governmental) should be obtained before
handling the animals. For experiments involving animals, please adhere
to the ARRIVE guidelines21. All procedures involving axolotls in our
laboratory are performed according to the animal and biological safety
level 2 (S2) license (24D-9168.11-9-2006-1 and 55-8811.72/34-16) issued
by Regierungspräsidium Dresden and Sächsisches Staatsministerium für
Umwelt und Landwirtschaft (SMUL), respectively. The ethical approval
was obtained from the Landesdirektion for animal welfare for the State of
Saxony, Germany.
532 | VOL.9 NO.3 | 2014 | nature protocols
worth keeping such animals for breeding and checking specificity
of gene expression in the F1 animals.
Our impression with Tol2 transgenesis is that the frequency of
ectopic expression is lower, presumably due to the fact that a higher
proportion (if not all) of the injected DNA is integrated into the
genome at the F0 stage. We had to carefully titrate the concentra-
tion of DNA in the Tol2 injections, presumably because of the high
rate of incorporation of DNA into the genome with this method.
We have generated germline loxP reporter and inducible Cre
driver axolotl lines11. The loxP reporter has the following DNA
construct integrated into its genome: CAGGs:loxP-eGFP-STOP-
loxP-Cherry-STOP. ‘CAGGs’ is a ubiquitous promoter (driving
expression in all cell types). Therefore, all the cells of this animal
express eGFP and no Cherry expression is observed because of
the STOP signal (3× polyadenylation) after the eGFP gene. The
‘eGFP-STOP’ DNA sequence is cloned in between loxP sites for
Cre-mediated recombination.
The Cre driver has the following genotype: CAGGs:ERT2-Cre-
ERT2-T2A-nuc-eGFP-STOP. ‘CAGGs’ drives the expression of Cre
recombinase fused with two mutated estrogen-binding domains
(ERT2−Cre-ERT2) followed by the nuclear localized eGFP (nuc-
eGFP) in each cell. The fusion of mutated estrogen-binding
domains with Cre allows us to induce Cre recombination with
tamoxifen. This facilitates a spatial and temporal control of Cre-
mediated recombination in germline transgenic axolotls.
As described below, a tamoxifen-inducible Cre driver line
is mated with a loxP-reporter axolotl line to produce double-
transgenic animals (CAGGs:ERT2-Cre-ERT2-T2A-nuc-eGFP/+;
CAGGs:loxP-eGFP-STOP-loxP-Cherry-STOP/+). These dou-
ble-transgenic progeny are grown to a desired size and treated
with tamoxifen to induce Cre-mediated recombination. Upon
tamoxifen-induced Cre-mediated recombination, the eGFP cas-
sette is excised out of the genome and the cells start expressing the
Cherry fluorescent protein (CAGGs:loxP-Cherry-STOP).
Tamoxifen treatment is performed either by an i.p. injec-
tion or by adding tamoxifen to the rearing water. In our hands,
both methods result in the same efficiency of Cre-mediated
recombination. However, it is recommended to use water-based
induction for smaller animals (1–4 cm in length from snout
to tail tip), as they are fragile, so it is tedious and not feasible
to inject them.
• Artemia (http://www.ambystoma.org/education/guide-to-axolotl-husbandry/
simple-brine-shrimp-hatchery)
• Ficoll PM 400 (Sigma-Aldrich, cat. no. 46327-500G-F)
• Benzocaine, to anesthetize the animals (Sigma-Aldrich, cat. no. E1501)
• NaCl (Merck, cat. no. 106404)
• MgCl2 (Merck, cat. no. 105833)
• KOH (Merck, cat. no. 814353)
• EDTA (Merck, cat. no. 324506)
• HEPES (Merck, cat. no. 391338)
• KCl (Merck, cat. no. 104936)
• CaCl2 (Merck, cat. no. 102382)
• MgSO4 (Merck, cat. no. 105886)
• HCl, fuming, 37% (vol/vol) (Merck, cat. no. 100317)
• Ethanol, absolute, to rinse the eggs (VWR, cat. no. 20821-330)

• Tris (Carl Roth, cat. no. 4855.2)
• Penicillin-streptomycin (PAA, cat. no. P11-010)
• l-Thyroxine (Sigma, cat. no. T2376)
• DMSO (Sigma, cat. no. D2650)
• 4-Hydroxytamoxifen (Sigma, cat. no. H7904)
• BspEI (New England Biolabs (NEB), cat. no. R0540)
• I-SceI enzyme and I-SceI enzyme buffer (NEB, cat. no. R0694)
• Plasmids: pBSII-SK-mTol2-MCS and T7-TPase (available on request from
the authors)
• RNaseZap (Ambion-Life Technologies, cat. no. AM9780)
• Qiagen plasmid maxi kit (Qiagen, cat. no. 12163)
• Fast Green FCF dye (Sigma, cat. no. 7252)
• UltraPure DNase-/RNase-free distilled water (Life Technologies,
cat. no. 10977-023)
• DPBS (Life Technologies, cat. no. 14190)
• DNA-grade agarose (Life Technologies, cat. no. 75000-500)
• Agar (SERVA, cat. no. 11393.04)
EQUIPMENT
• Scalpels to scrape the eggs from the leaves (Cutfix; Medical,
cat. no. 5518075)
• Containers for collecting eggs and for keeping the animals (VWR,
cat. no. 216-3416)
• Cups for keeping the small animals, 250 ml Becher (Sarstedt,
© 2014 Nature America, Inc. All rights reserved.
cat. no. 75 560)
• Sieve for rinsing eggs (Carl Roth, cat. no. 8098.1)
• Fish nets (http://www.zajac.de/): 20 cm (cat. no. AQ161); 10 cm
(cat. no. AQ164); 8 cm (cat. no. AX100165)
• Three pairs of forceps: two to dejelly the eggs and one to break the needle
for injection (F.S.T, cat. no. 11 295-51)
• Petri dishes to collect eggs (Greiner Bio-One): 145 mm, cat. no. 639 102;
94 mm, cat. no. 633 180; 60 mm, cat. no. 628 102; 35 mm, cat. no. 627 102
• DNase-/RNase-free sterile pipette filter tips (Fisher Scientific): 0.1–10 µl,
cat. no. 02-707-439; 2–20 µl, cat. no. 02-707-432; 10–100 µl, cat. no. 02-707-
431;20–200 µl, cat. no. 02-707-430
• Plates (24 wells) to keep the embryos on the first week (Nunc, cat. no. 142475)
• Sterile plastic transfer pipettes (2 ml) to transfer the embryos (Sarstedt,
cat. no. 86.1171.001)
• PCR tubes, 0.2 ml (Bio-Rad, cat. no. TWI-0201)
• Slide with micrometric scale, 0.1 mm to determine the volume of the
injection droplet (Bresser)
• Mold for making injection plates (custom manufactured at the MPI-CBG
machine workshop; dimensions shown in Fig. 1a)
• Microscope (Olympus SZX10)
• Light source (Olympus KL 1500 compact)
• PV830 pneumatic Pico pump (WPI)
• Micromanipulator, Narishige MN-153 (Science Products)
• Magnetic stand GJ-1 (Science Products)
• Steel plate IP for use with magnetic stand (Science Products)
• M/F pressure-monitoring/injection line, 5 feet (Argon Medical Devices,
cat. no. 040105005A)
• Microelectrode holder (WPI, cat. no. MPH6S10)
• Injection needles: borosilicate glass capillaries GC100F-15, 1.00 mm outer
diameter × 0.58 mm inner diameter (Harvard Apparatus, cat. no. 30-0020)
• Flaming/Brown micropipette puller P-97 (Sutter Instrument Company)
• Box filament FB255B, 2.5 × 2.5 mm (several different filaments come with
the puller)
• mMESSAGE mMACHINE T7 ultra kit (Ambion-Life Technologies,
cat. no. AM1345)
• Gas-tight Hamilton syringe (Hamilton, cat. no. 1702)
• Disposable needle, 30 G, 1/2 inch, 0.3 × 13 mm (BD, cat. no. 305771)
PROCEDURE
Axolotl mating and egg collection ● TIMING 3–4 d
1| Before the mating process, keep a female axolotl in isolation in a single-housing tank for 2 d without water circulation.
The ideal temperature of water should be 18 °C.
2| Put the male in a separate tank (mating tank) for (the same) 2 d.
3| Put the female into the mating tank with the male after the 2-d isolation is over.
protocol
REAGENT SETUP
Marc’s modified Ringer’s solution (MMR), 10× To prepare 2 liters of
10× MMR, mix 400 ml of 5 M NaCl, 40 ml of 1 M KCl, 20 ml of 1 M MgCl2,
40 ml of 1 M CaCl2, 4 ml of 0.5 M EDTA and 100 ml of 1 M HEPES. Bring
the volume to 1.5 liters with deionized water. Adjust the pH to 7.8 with 10 M
KOH, and then fill up to 2 liters with deionized water. Autoclave the solution
and store it at room temperature for up to 6 months.
MMR (1×)/pen-strep To prepare 1 liter of 1× MMR/pen-strep, mix 100 ml
of 10× MMR with 13 ml of penicillin-streptomycin and bring the volume to
1 liter with deionized water. Filter the solution, sterilize it and store it at room
temperature for up to 2 months.
MMR (1×)/20% (wt/vol) Ficoll/pen-strep To prepare 250 ml, mix 25 ml of
10× MMR, 50 g of Ficoll and 7 ml of penicillin-streptomycin. Bring the
volume to 250 ml with deionized water. Filter the solution, sterilize it and
store it at 4 °C for up to 2 months.
MMR (0.1×)/5% (wt/vol) Ficoll/pen-strep To prepare 1,000 ml of solution,
mix 10 ml of 19× MMR, 50 g of Ficoll and 13 ml of penicillin-streptomycin.
Make up the volume to 1,000 ml with deionized water. Filter the solution,
sterilize it and store it at 4 °C for up to 2 months.
MMR (0.1×)/pen-strep To prepare 2 liters of solution, mix 20 ml of 10×
MMR and 25 ml of penicillin-streptomycin. Make up the volume to 2 liters
with deionized water. Filter the solution, sterilize it and store it at room
temperature for up to 2 months.
Holtfreter’s solution, 400% (wt/vol) concentration To prepare 10 liters of
Holtfreter’s solution, mix 2.875 g of KCl, 5.36 g of CaCl2·2 H2O, 11.125 g of
MgSO4·7 H2O and 158.4 g of NaCl. Make up the volume to 10 liters with
distilled water. Store the solution at room temperature for up to 6 months.
TBS, 10 × To prepare 1,000 ml of 10× TBS, add 24.2 g of Tris and 90 g of
NaCl in 990 ml of distilled water. Mix well with a stir bar and adjust the pH
to 8.0 by adding 10 ml of HCl (37% vol/vol). Store the solution at room
temperature for up to 6 months.
Benzocaine 10% (wt/vol) To prepare 500 ml of 10% (wt/vol) benzocaine,
mix 50 g of benzocaine in 500 ml of 100% ethanol. Store the solution at
room temperature for up to 12 months.
Anesthetic for axolotls, 0.03% (vol/vol) benzocaine To prepare 10 liters
of anesthetic, mix 500 ml of 10× TBS, 500 ml of 400% Holtfreter’s solu-
tion and 30 ml of 10% (wt/vol) benzocaine. Bring the volume to 10 liters
with distilled water and store the solution at room temperature for up to
6 months.
4-hydroxytamoxifen (4-OH tamoxifen) solution For i.p. injections
(10 mg/ml), dissolve 100 mg of 4-hydroxytamoxifen in 10 ml of DMSO.
Divide the prepared solution into aliquots and freeze them at −80 °C for up
to 6 months. Avoid freeze-thaw cycles and protect the solution from light.
For water-based induction (20 mM), dissolve 77.4 mg of 4-hydroxytamoxifen
in 10 ml of DMSO. Divide the solution into aliquots and freeze them at
−80 °C for up to 6 months. Avoid freeze-thaw cycles and protect the solution
from light. ! CAUTION Weigh 4-OH tamoxifen powder in a hood, and avoid
inhalation and contact with skin.
Thyroxine solution, 10 mg/ml Dissolve 10 mg of thyroxine in 1 ml of
DMSO. Add 9 ml of DMSO to this stock solution to obtain 1 µg/µl of
working solution. Divide the solution into aliquots and freeze them at −80 °C
for up to 6 months. Avoid freeze-thaw cycles and protect the solution from
light. ! CAUTION Weigh thyroxine powder in a hood and avoid inhalation
and contact with skin.
Fast Green FCF, 5× solution Dissolve 12.5 mg of Fast Green FCF powder in
10 ml of DPBS. Store the solution at 4 °C for up to 6 months. Use it at 1× for
injection with 4-OH tamoxifen.
nature protocols | VOL.9 NO.3 | 2014 | 533
 protocol

4| Cover the mating tank and stop the flow of fresh water to minimize the dilution and washing away of pheromones.
If there is no automated water temperature control, keep the mating tank in a room in which the temperature does not rise
above 18 °C.

5| Place some artificial plastic leaves in the mating tank so the females can lay eggs on them (Fig. 1c).

6| Although they are not strictly necessary, some stones/small rocks can also be placed in the mating tank so that the
males can lay spermatophores on them.

7| After the mating ritual, the male lays multiple spermatophores (Fig. 1d, Supplementary Video 4). There is no specific
time for the males to lay spermatophores. In our colony, males sometimes lay spermatophores between 5 and 30 min after
the mating ritual. In more than half of the cases, the spermatophores are seen only after the change in day/light cycle.
? TROUBLESHOOTING

8| The female will pick up the spermatophores through her cloaca. If the female picks up the spermatophore during the
night, one can see the leftover jelly part of the spermatophore, whereas the bright white tips (containing the actual sperm)
will be gone (Fig. 1d). If the female picks up the spermatophore, her cloacal opening will increase in size, which is a good
indication of a successful mating (Supplementary Fig. 2c,d).
© 2014 Nature America, Inc. All rights reserved.

9| Approximately 18–24 h after the female picks up the spermatophores, she will start laying one-cell–stage fertilized eggs
(Supplementary Video 4). Leave the male inside the mating tank while the female is laying eggs.
? TROUBLESHOOTING

10| The female will hold the artificial leaves with her hind limbs and lay eggs on them. Carefully remove the artificial plastic
leaves from the tank without disturbing the female if she is still laying eggs.

11| Use a scalpel to collect the eggs from the artificial plastic leaves underwater (Fig. 1e, Supplementary Video 4).

12| If the female is disturbed while laying eggs, she may pause for a few hours and a timely collection of one-cell–stage
eggs will not be feasible. For transgenics, only one cell–stage eggs can be used.

13| The female axolotl usually starts laying eggs within 18–24 h after picking up the spermatophores (Step 9). If these one
cell–stage fertilized eggs are laid at a time when it is not feasible for the researcher to inject them, then put the female in
a covered small tub with cold water (~11 °C) with artificial plastic leaves and place the whole tub in a refrigerator at 11 °C.
Reducing the temperature slows both egg laying and embryo division. The female can be left at 11 °C overnight if necessary.

14| If the mating is done for other purposes (e.g., for checking a founder animal for germline transmission), for which
one-cell–stage eggs are not required, remove the female as soon as she starts laying eggs and put her in a covered small tub
with artificial plastic leaves until she is done laying eggs.
! CAUTION The regular tap water (in Dresden, Germany) is optimal for growth and rearing of axolotls. In other places, it is
important to use water that is dechlorinated by overnight evaporation, chemical conditioning and/or carbon filtering.

Collection of one-cell–stage eggs (embryos) ● TIMING 30–45 min

15| Collect the eggs by scraping them from the leaves with a scalpel; put them into a container with fresh tap water (Fig. 1e,
Supplementary Video 4). An average of 300–500 eggs can be obtained from a single spawn. In our colony, the largest
single spawn was 702 eggs.

16| Cut the tip of a plastic transfer pipette to make a wide bore (3–5 mm). By using the pipette, pick up the eggs and place
them in a metal sieve. As the eggs are connected by a jelly coat, masses of eggs will be picked up together.

17| Rinse the eggs with 70% (vol/vol) ethanol for ~20 s to sterilize them, and then wash them thoroughly with sterile water
(Supplementary Video 4).
 CRITICAL STEP It is important to use water that was dechlorinated by overnight evaporation, chemical conditioning and/
or carbon filtering.

18| With the help of the wide-bore transfer pipette, place the eggs in a Petri dish containing sterile 1× MMR/pen-strep solution.

534 | VOL.9 NO.3 | 2014 | nature protocols

 protocol

Box 2 | I-SceI–mediated transgenesis ● TIMING 30–45 min per 100 eggs

We have previously described a protocol using the I-SceI meganuclease for the generation of transgenic axolotls15, which was further
adopted to generate transgenic newts8. Clone the expression cassette in the MCS of the plasmid shown in Figure 1f such that the
I-SceI nuclease sites surround the cloned expression cassette. The resulting DNA construct can be used for both I-SceI– and Tol2-
mediated transgenics. The standard injection mix for generating transgenic axolotls (using I-SceI meganuclease) is 0.5 ng of DNA per
embryo in a 5-nl injection volume. Prepare the injection mix as follows:
   1 µg DNA
   1 µl 10× I-SceI buffer
   2 µl I-SceI 5,000 U/ml
   Fill up to 10 µl with DNase-/RNase-free H2O
Fill up the pulled glass capillary/needle (Step 36) with this mixture and inject the eggs as described in Steps 41–48.

19| Under an Olympus SZX10 or similar microscope, dejelly the eggs manually by using sharp tweezers (Supplementary Video 4).
Dejelly only the healthy eggs with a clearly visible, uniform dark-brown animal pole for injections (Supplementary Fig. 2a,b).

20| Transfer the dejellied eggs with a sterile transfer pipette into a fresh Petri dish containing sterile 1× MMR/pen-strep
© 2014 Nature America, Inc. All rights reserved.

solution.
 PAUSE POINT If the eggs are freshly laid, the Petri dish containing the eggs can be stored at 4 °C for 2–3 h, as the first cell
division is between 6 and 7 h after egg-laying. It is recommended to inject the eggs with DNA solution as soon as possible.

Transgenesis with the mini Tol2 transposon: cloning and prepping plasmids with mini Tol2 sites ● TIMING 5–7 d
 CRITICAL For I-SceI-mediated transgenesis, follow Box 2.

21| Clone the transgene of interest expression cassette in the plasmid pBSII-SK-mTol2-MCS (or in a similar plasmid) such
that the mini Tol2-recognition sequences flank the expression cassette (Fig. 1f). It is necessary to include a fluorescent
reporter gene as a means of visual screening of transgenic founder animals.

22| Purify the plasmid DNA with the Qiagen plasmid maxiprep kit according to the manufacturer’s instructions.

23| Suspend the purified DNA pellet in 200–300 µl of DNase-/RNase-free water and keep it overnight at 4 °C to dissolve.
On the next day, mix by gently pipetting the dissolved DNA and dilute it to a concentration of 1 µg/µl in DNase-/RNase-free
water.

24| Verify the integrity of plasmid DNA by restriction analysis and sequencing of the plasmid DNA to make sure that there
are no mutations in the cloned cassette (promoter, gene of interest and polyadenylation signal). This is achieved by design-
ing multiple primers for sequencing that cover the entire cloned cassette.

25| Make small aliquots of the plasmid and store the DNA at −20 °C until use.
 PAUSE POINT Plasmid DNA can be stored indefinitely at −20 °C.

In vitro transcription of the Tol2 transposase mRNA ● TIMING 2 d

26| Linearize 5–10 µg of T7-TPase plasmid DNA with BspEI restriction enzyme with an overnight digestion reaction at 37 °C.
For this, mix 5–10 µg of T7-Tpase plasmid DNA, 10 µl of NEBuffer 3.1, 2 µl of BspEI enzyme and make the volume up to
100 µl with DNase-/RNase-free H2O.

27| Run 5–10% of the digested DNA in a 1% (wt/vol) agarose gel to make sure that all of the plasmid has been linearized.
Linearized plasmid will show a single sharp band of 5.3 kb. As a control, 500 ng of undigested plasmid can be run alongside
the linearized plasmid.

28| Transcribe the Tol2 RNA in vitro by using the mMESSAGE mMACHINE T7 ultra kit according to the manufacturer’s instructions.
The protocol will yield an RNA product that is further stabilized by the tailing of additional polyA tail according to the
instruction manual of the mMESSAGE mMACHINE T7 ultra kit. Save 2.5 µl of the RNA before proceeding to polyA tailing.
 CRITICAL STEP The in vitro transcription and all RNA handling should be performed in RNase-free conditions. Wipe all
bench surface and pipettes with RNaseZap or equivalent reagents. Use filter DNase-/RNase-free pipette tips. Keep the
in vitro transcription reaction and components on ice unless stated otherwise in the mMESSAGE mMACHINE T7 ultra kit’s
instruction manual.
nature protocols | VOL.9 NO.3 | 2014 | 535
 protocol

29| Precipitate the RNA by using the LiCl method according to the instruction manual of the mMESSAGE mMACHINE T7 Ultra
kit. Dissolve the RNA pellet in 20 µl of DNase-/RNase-free water.

30| Measure the concentration of RNA and adjust the concentration to 500 ng/µl by using DNase-/RNase-free water.
We routinely obtain 20–25 µg of RNA starting with 1 µg of linearized template DNA for in vitro transcription.

31| Run 500 ng of the RNA on 1% (wt/vol) agarose gel alongside 2.5 µl of the RNA before polyA tailing (Step 28). Both
samples should run as single sharp bands and not as smears. The band of the RNA after polyA tailing should be bigger in size
than that of the RNA before polyA tailing, confirming the addition of a polyA tail.

32| Make aliquots (on ice) of 4.1 µl of the 500 ng/µl stock in 0.2-ml tubes and store them at −80 °C.
 PAUSE POINT In our experience, the RNA is stable for up to 2 years when stored at −80 °C and handled properly.

Injection plate preparation ● TIMING 15–30 min

33| Prepare a 2% (wt/vol) agar gel solution with water and pour ~10 ml of the solution into a 100-mm Petri dish.

34| Place the plastic mold (Fig. 1a) upside down in the 2% (wt/vol) agar solution and let the gel solidify at room
temperature. This creates channels in the gel for holding the eggs in place for injection.
© 2014 Nature America, Inc. All rights reserved.

35| Remove the mold and place the Petri dish at 4 °C until use.
 PAUSE POINT Multiple injection plates can be made at a time and then sealed with Parafilm and stored at 4 °C for up to 2 weeks.

Injection needle preparation ● TIMING 15–30 min

36| Pull the borosilicate glass capillaries with the P-97 Flaming/Brown micropipette puller according to the following parameters,
and then stock them for future use (Supplementary Fig. 2f): P = 500; heat = 530; pull = 100; velocity = 120; time = 150.

Injection of DNA or RNA-DNA mix ● TIMING 30–45 min per 100 eggs
37| Fill the injection plate (Step 35) with 1× MMR/20% Ficoll/pen-strep solution.

38| Transfer the dejellied eggs (Step 20) into the channels of the injection plate (with an average of 20 eggs per channel) as
shown in Figure 1b and in Supplementary Video 5. The eggs should be covered with the 1× MMR/20% Ficoll/pen-strep solution.

39| Prepare the mix of transgene expression plasmid (Step 25) and Tol2 transposase RNA (Step 32) to be injected on ice.
The ideal plasmid and RNA amounts in a 5-nl injection volume per egg are 0.05 ng of plasmid DNA and 0.25 ng of Tol2 RNA.
As a control for the efficiency of transgenesis with and without the Tol2 transposase, inject only DNA into some eggs without
the Tol2 in vitro–transcribed RNA. Keep the reaction mixture on ice and proceed to the next step as soon as possible.
 CRITICAL STEP To obtain the highest efficiency of the strong category of transgenic animals (see Experimental design), we
recommend titrating the necessary amounts of RNA and DNA for each new plasmid size being used (see Experimental design).
The amounts stated above are for a plasmid of ~8,000 bp in size.
 CRITICAL STEP If multiple DNA/RNA ratios or different plasmids are injected in one injection sitting, please use a separate
injection plate for each condition. Furthermore, clean the needle holder and microscope with 70% (vol/vol) ethanol, followed
by distilled water, to avoid cross-contamination between injections.

40| Fill up the needle (pulled glass capillary, Step 36) with the DNA/RNA injection mix (Supplementary Video 5).

41| Fit the needle to the micromanipulator and pneumatic injection pump.

42| Measure the drop size by using a slide with a micrometer scale (Supplementary Video 5). Adjust the droplet size to 5 nl
by breaking the tip of the glass needle with forceps under the Olympus SZX10 or similar microscope.

43| Inject the eggs in their animal pole region (dark brown region) such that the needle is inserted into one quarter of the
whole egg (half of the animal pole; Supplementary Video 5).

44| Transfer the injected eggs into a new Petri dish containing fresh 1× MMR/20% Ficoll/pen-strep solution and put the lid
on the Petri dish. Leave it at room temperature for 2 h. As a control for egg quality and development of embryos, keep
some dejellied, un-injected eggs (10–20 eggs per spawn) in a new Petri dish containing fresh 1× MMR/20% Ficoll/pen-strep
solution.

536 | VOL.9 NO.3 | 2014 | nature protocols

 protocol

45| Use sterile transfer pipettes to transfer the injected and control uninjected eggs (one by one) into a 145-mm (injected
eggs) and 100-mm (control eggs) Petri dish containing 0.1× MMR/5% Ficoll/pen-strep solution. Do not put more than 100
injected eggs in the 145-mm dish. Put the lid of the Petri dish on it and leave the eggs at room temperature for 24 h on the
bench. Do not move or disturb the plate after placing the eggs.

46| Observe the eggs under the Olympus SZX10 or a similar microscope and transfer the healthy eggs (Supplementary
Fig. 2a,b) into a sterile 24-well plate, one embryo per well, containing 0.1× MMR/pen-strep solution. Leave the eggs in
this plate for 7 d on a benchtop with a maximum temperature of 24 °C. No change of solution is required for 7–8 d.

47| After 7–8 d, transfer the healthy developed embryos into a Petri dish with fresh 0.1× MMR/pen-strep solution; the dish
should be kept on the benchtop with a maximum temperature of 24 °C. The health of injected embryos can be ascertained
by comparing them morphologically with the uninjected control embryos. Please also refer to (http://www.ambystoma.org/
education/embryo-staging-series) for the various developmental stages of axolotls.

48| The larvae will hatch and start swimming within 2–3 d.

Screening of larvae for transgene expression ● TIMING 60 min per 100 embryos
49| The larvae are ready to be screened as soon as they hatch and start swimming (Fig. 1g).
© 2014 Nature America, Inc. All rights reserved.

50| Dilute 0.03% (wt/vol) benzocaine (anesthetic) to 0.01% with distilled water.

51| Cut the tip of a transfer pipette such that it can be used to pick up freshly hatched larvae.

52| Place the wide-bore transfer pipette in such a way that the larvae can swim directly into the wide-bore opening.

53| Once the larvae are inside the pipette, place the larvae in 0.01% (wt/vol) benzocaine solution (by gently squeezing the
bulb of the transfer pipette) in a Petri dish and wait until they are anesthetized.

54| Gently pick one larvae with a wide-bore transfer pipette and place it in a Petri dish (containing the anesthetic) under
the fluorescence microscope to screen for transgene expression.

55| Transfer the transgenic larvae to small plastic cups or tubs and feed them daily with live Artemia, starting 12–14 d after
injection (Step 43).

Tamoxifen-induced Cre-mediated recombination ● TIMING up to 1 d

56| For 4-OH tamoxifen induction by i.p. injections, use option A. For water-based 4-OH tamoxifen induction, use option B.
The earliest recombination event is visible (depending on your readout system) 5–7 d after 4-OH tamoxifen treatment, and
transgene expression is stabilized after 2 weeks of 4-OH tamoxifen treatment and/or Cre recombination.
(A) 4-OH tamoxifen induction by i.p. injection ● TIMING 60 min per 20 animals
(i) Wet a tissue paper or Kimwipe in 0.01% (wt/vol) benzocaine and place it in a Petri dish.
(ii) Anesthetize animals >4 cm in length in 0.03% (wt/vol) benzocaine.
 CRITICAL STEP Animals measuring ≤4 cm in length should be induced in water.
(iii) Weigh the animal and determine the amount of 4-OH tamoxifen injection volume necessary, calculating 5 µl per gram
of body weight.
(iv) Place the animal on a wet tissue inside the Petri dish.
(v) Thaw the 10 mg/ml 4-OH tamoxifen stock (from −80 °C) and add Fast Green FCF to it to obtain 1× dye solution.
(vi) Fill up the Hamilton syringe fitted with a 30-G, 1,2½-inch, 0.3 × 13 mm needle with the desired volume of 4-OH
tamoxifen (according to the weight of the animal).
(vii) Intraperitoneally inject the required volume and cover the animals in wet tissue paper for 20–30 min before returning
them to fresh water (Fig. 1h).
(B) Water-based tamoxifen induction ● TIMING 1 d
(i) Thaw the 20 mM stock of 4-OH tamoxifen (from −80 °C).
(ii) Measure the amount of water in the rearing tank or tub.
 CRITICAL STEP It is important to use water that was dechlorinated by overnight evaporation, chemical conditioning
and/or carbon filtering.
(iii) Add 4-OH tamoxifen to a final concentration of 2 µM in the rearing water for animals up to 4 cm in length (from nose
to tail tip), and a 5 µM final concentration for animals larger than that.
 CRITICAL STEP 4-OH Tamoxifen is light sensitive; hence, the containers with 4-OH tamoxifen should not be exposed
to light.

nature protocols | VOL.9 NO.3 | 2014 | 537

 protocol

(iv) Change the water after 16 h of exposure to regular water without 4-OH tamoxifen.
? TROUBLESHOOTING

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.

Table 1 | Troubleshooting table.

Step Problem Possible reason Solution

7 No spermatophores Water temperature is not adequate Provide a fresh flow of water for 2–3 h, as fresh water can
are seen 24 h after stimulate the animals to mate. If you are setting up mating
setting up the mating in a nonautomated system, change the water to a colder
temperature of 15–16 °C

The mating tank is not covered or Cover the mating tank and do not disturb the animals
the animals are disturbed after the
mating is set up

The male is old or has mated within Replace the male or female. We recommend using animals that
© 2014 Nature America, Inc. All rights reserved.

the last 4 weeks are not older than 3–4 years for mating

9 No eggs are laid The female did not pick up the Set up a mating with a new pair
spermatophores

The female had spawn within the Keep track of mating dates of the animals
last 8 weeks

The daylight cycle is not optimal in Reduce the light cycle to 8 h of light per day and leave this
the colony cycle in place for 1 week. Increase the amount of daylight by
15 min on both the beginning and the end of the day (30 min
total increase per day) until reaching 12 h of light per day

56 No Cre-mediated The ER-Cre-ER gene is under the Multiple rounds of 4-OH tamoxifen can be given for a weak
recombination control of a weak promoter promoter or enhancer driving the ER-Cre-ER gene. The 4-OH
tamoxifen treatment can be repeated every day for 16 h with
fresh 4-OH tamoxifen-containing water. In our laboratory,
we have induced (in water) axolotls for 5 consecutive days
without observing any toxic effects on growth or regeneration
of the animals

The 4-OH tamoxifen stock is old or Avoid multiple freeze-thaw cycles of stored aliquots. Protect
not properly stored aliquots from light

The injected 4-OH tamoxifen did not Keep the 4-OH tamoxifen–injected animals in a hydration
diffuse to all tissues or cells of the chamber for at least 20–30 minutes before returning them to
body fresh water

Dead animals The batch of 4-OH tamoxifen is toxic Try a new batch or lot of 4-OH tamoxifen, as some lots give
better results than others

Box 1, No metamorphoses The thyroxine stock is not made Prepare a new exact thyroxine stock
step 10 or animal death properly Reduce the amount of thyroxine injected into the animal to
1.0–1.2 µg per gram of body weight of the animal

DMSO used is not pure Only use cell culture–grade DMSO to make thyroxine solutions

● TIMING
Steps 1–14, axolotl mating and egg collection: 3–4 d
Steps 15–20, collection of one-cell–stage eggs (embryos): 30–45 min
Steps 21–25, cloning and prepping of plasmids with miniTol2 sites: 5–7 d
Steps 26–32, in vitro transcription of the Tol2 transposase mRNA: 2 d

538 | VOL.9 NO.3 | 2014 | nature protocols

 protocol

Steps 33–35, injection plate preparation: 15–30 min

Step 36, injection needle preparation: 15–30 min
Steps 37–48, injection of DNA or RNA-DNA mix: 30–45 min per 100 eggs
Steps 49–55, screening of larvae for transgene expression based on fluorescence: 60 min per 100 embryos
Step 56, 4-OH tamoxifen-induced Cre-mediated recombination: up to 1 d
Box 1, metamorphosing axolotls: 60 min per 20 animals
Box 2, I-SceI–mediated transgenesis: 30–45 min per 100 eggs

ANTICIPATED RESULTS
In our hands, Tol2-mediated transgenesis has resulted in ~30% of the offspring being of the strong category of transgenic
animals that can either be grown to sexual maturity or used at a desired size for experiments without waiting for 9–12
months to obtain the F1 generation.
We suggest that a small-scale pilot experiment be done for each DNA construct of different size to figure out the optimal
DNA-to-RNA ratio required to achieve ‘strong’ healthy transgenic larvae. Once the optimal DNA-to-RNA ratio for the construct
is determined, inject DNA in some eggs with and without the Tol2 in vitro–transcribed RNA to check the efficiency of
transgenesis with and without Tol2 transposase.
The table below, compiling the results of three independent transgene injections/experiments, shows a titration of a
transgene expression plasmid (~8,000 bp in size) co-injected with Tol2 in vitro–transcribed RNA.
© 2014 Nature America, Inc. All rights reserved.

Category of expression
Amount of Amount of Tol2 Embryos Strong Medium Weak
plasmid (ng) mRNA (ng) injected Survivors (% survivors) (% survivors) (% survivors)

0.05 0 44 35 0 6 (17%) 29 (83%)

0.15 0 42 9 0 1 (11%) 8 (89%)

0.2 0 35 2 0 0 2 (100%)

0.3 0 38 8 0 1 (13%) 7 (87%)

0.025 0.125 118 97 21 (22%) 8 (8%) 68 (70%)

0.05 0.25 379 220 76 (35%) 49 (22%) 95 (43%)

0.05 0.375 127 43 2 (5%) 14 (32%) 27 (63%)

0.05 0.5 66 37 3 (8%) 8 (22%) 26 (70%)

0.075 0.25 521 240 10 (4%) 82 (34%) 148 (62%)

0.15 0.25 66 4 0 2 (50%) 2 (50%)

0.2 0.25 92 5 0 4 (80%) 1 (20%)

0.3 0.25 35 0 0 0 0

Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments This manuscript would not have been possible without the
dedicated hard work and commitment from our animal caretakers: H. Andreas,
J. Michling, S. Mögel, C. Junghans, M. Armstead and B. Gruhl. We thank
S. Borland (former Axolotl colony at Indiana University) for advice on the
day/light cycle for mating troubleshooting, and K. Boes and S. Hermann (Center
for Regenerative Therapies Dresden) for help in obtaining images and movies for
this manuscript. This work was supported by funds from the VolkswagenStiftung,
DFG grant nos. TA274/3-1, TA274/3-2, TA274/5-1, a European Research Council
Advanced Investigator Grant, central funds of the MPI-CBG and the DFG Center
for Regenerative Therapies.
AUTHOR CONTRIBUTIONS S.K., P.M., H.A., V.K., M.S., T.S.-G., K.C. and E.M.T.
contributed to the various protocols described here. E.M.T. provided financial
support and supervised the lab personnel. S.K. and E.M.T. designed the
experiments, analyzed the data and wrote the final manuscript.
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.
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