Download Congenic method in the chick limb buds by electroporation

Develop. Growth Differ. (2008) 50, 459–465
doi: 10.1111/j.1440-169x.2008.01054.x
Review
Blackwell Publishing Asia
Congenic method in the chick limb buds by electroporation
Takayuki Suzuki* and Toshihiko Ogura
Institute of Development, Aging and Cancer (IDAC), Tohoku University, Seiryo-cho 4-1, Aoba-ku, Sendai, 980-8575, Japan
Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an
intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes.
In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific
expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the
expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant
embryonic patterning. Finally, useful trouble-shooting techniques are described.
Key words: chick embryo, electroporation, limb buds, limb field.
Introduction
The developing limb bud is an excellent model with
which to study molecular mechanisms of embryonic
patterning and tissue differentiation in vertebrates. By
exploiting both classical and modern embryological
methods, combined with novel techniques in molecular
biology, data regarding limb development is rapidly
accumulating. Classical embryological experiments,
such as apical ectodermal ridge (AER) removal from
the chick limb bud, revealed the presence of a key
signaling pathway, and ultimately resulted in the discovery of the fibroblast growth factor (FGF) cascade.
FGF interacts with the zone of polarizing activity (ZPA)
(Saunders & Gasseling 1968), a region located at the
posterior side of the limb bud specifying posterior limb
bud identity, discovered by another classic embryological
experiment, the anterior implantation of a tissue graft
from the posterior chick limb bud. Thus two opposite
approaches, loss-of-function and gain-of-function
experiments, are necessary to dissect the molecular
nature of these signal transduction pathways.
In the mouse, targeted gene disruption is a powerful
loss-of-function approach. For example, knocking-out
FGF10 resulted in the total loss of limb buds in the
*Author to whom all correspondence should be addressed.
Email: [email protected]
Received 14 March 2008; revised 4 April 2008; accepted 1 May
2008.
© 2008 The Authors
Journal compilation © 2008 Japanese Society of
Developmental Biologists
mouse (Sekine et al. 1999). Since the first report of
gene targeting was published (Thomas & Capecchi
1987), this technique has been an invaluable tool for
developmental biologists, providing essential insights
into the developmental function of embryonic genes.
Nonetheless, knockout mice often show lethality during
early stages of development, making it difficult to study
gene function during organogenesis at later stages,
such as limb development. To overcome this problem,
the conditional knock-out technique was developed
using tissue-specific expression of the Cre recombinase;
today this approach is limited only by the availability of
tissue-specific promoters.
In contrast to the genetic approaches developed in
mice, two different approaches have been developed
in chick models to introduce transgenes in a spatially
and temporally controlled manner; one using retroviruses,
the other, electroporation. Electroporation has several
advantages over retrovirus-mediated gene transfer,
including the ability to rapidly express cDNA after transient cell membrane disruption. Electroporation is also
able to introduce larger constructs into cells than
retrovirus-mediated techniques. However, retrovirusdependent methods feature the advantage of affording
researchers the ability to misexpress transgenes stably
even at later stages of limb development, making this
system versatile and applicable in a wide range of
scenarios in developmental biology.
In this review, we will describe in detail how to choose
expression vectors, as well as efficient methods for
using electroporation to introduce transgenes of interest
into chick limb buds.
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T. Suzuki and T. Ogura
Fig. 1. Anatomy of the limb field. (A) Forelimb field and hindlimb
field are shown in orange and green, respectively, at St. 14.
These are located on the lateral aspect of the somite, called the
lateral plate mesoderm (LPM). (B) A horizontal section of a St. 14
forelimb field is shown. Forelimb and hindlimb buds develop from
the dorsal side of the LPM, called the somatic LPM. There is
embryonic coelom between the somatic and splanchnic LPM.
D, dorsal side; V, ventral side.
Limb development in the chick embryo
Limb development begins as a small budding of the
lateral plate mesoderm (LPM) (Fig. 1A). In the chick
embryo, the forelimb bud starts to develop from a
restricted part of the LPM at the 15–20 somite level,
whereas the hindlimb develops at the 26–32 somite
level. The LPM consists of two different parts, namely
the somatic LPM on the dorsal side and the splanchnic
LPM on the ventral side (Fig. 1B), separated by an
embryonic space (coelom) (Fig. 1B). Both forelimb and
hindlimb develop from two distinct fields in the somatic
LPM, in which FGF10 starts to be expressed at St. 14
to induce cell proliferation and limb bud outgrowth.
Hence, one can identify the limb fields in embryos by
visualizing them with an FGF10 probe.
After the initiation of limb development, threedimensional axes are specified; namely, the proximal-distal
(PD), dorsal-ventral (DV) and anterior-posterior (AP) axes.
Differentiation of cells and subsequent pattern formation
proceeds in a temporally and spatially regulated
manner along these axes at later stages. For example,
mesenchymal cells condense to form cartilage, followed
by the migration of progenitor cells and their differentiation into muscles, tendons, and neurons, making
limb buds highly complex precursors to such intricate
structures as wings/hands (forelimb) and legs (hindlimb).
How to choose expression vectors for
electroporation
We use two different expression vectors for misexpression in embryos. One is pCAGGS (Niwa et al. 1991),
a transient expression vector constructed from an
enhancer of the human cytomegalovirus (CMV) immediate
early region and a chick beta-actin promoter. Another
vector is pRCASBP, an avian replication-competent
retrovirus vector (Morgan & Fekete 1996). pCAGGS
drives a more robust expression of transgenes compared
with pRCASBP, thus we observe misexpression of
transgenes within 3 h when pCAGGS expression is
monitored by enhanced green fluorescent protein
(EGFP). Nonetheless, expression is transient with maximal
transcription occurring 6–24 h after electroporation,
followed by a gradual decrease in the level of expression,
probably due to cell proliferation and concomitant dilution
of the electroporated plasmids. Therefore, the pCAGGS
vector is suitable to study the embryonic limb initiation
stage and limb bud stage (Takeuchi et al. 2003).
The utility of pRCASBP is quite distinct. Since
pRCASBP is a competent viral vector, transcripts from
electroporated DNA and their transcribed and translated protein products reconstitute infectious virions,
which then induce secondary infection in neighboring
cells when a competent chick host is used. This cycle
of infection results in a wide spread of transgene
expression at later stages. For this reason, initial
expression of transgene is relatively slow and weak
when compared with pCAGGS. However, an active
expansion of transgene expression is observed in tissues
where cells divide quickly, such as the progress zone
of limb buds, and we usually obtain expression in the
entire limb bud one day after electroporation. Therefore,
the RCASBP vector is suitable for study of later stages
of limb development such as skeletal patterning and
tissue differentiation (Takeuchi et al. 1999). Once the
RCASBP genome is integrated by the active virion,
expression is stable and lasts for the entire processes
of limb development. In contrast to pCAGGS, the
RCASBP vector has a limited capacity for transgene
inclusion (approximately 2.4 kb), since a longer insert
inhibits the incorporation of the viral RNA genome into
virions, and thereby prevents secondary infection and
spread of the expression domain. Such limitations are
also observed when virus-incompetent chick hosts are
used. However, because stable expression of transgenes can be observed in subpopulations of cells even
in this case, this indicates that electroporated RCASBP
plasmids can be integrated into the host genome.
In the typical protocol used for the pRCASBP
vector, infectious virions are produced from CEF (chick
embryonic fibroblast) cells after transfection, and then
concentrated to a titer sufficient for effective infection
after the injection of virions. Since the transgene is
expressed in CEF in this step of viral propagation, it is
not easy to obtain a high titer virus concentrate when
the transgene is toxic to CEF. We often encounter this
problem, especially when we use genes related to
© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists
Electroporation into chick limb buds
apoptosis and bone morphogenetic protein (BMP)
signaling. Nonetheless, by directly electroporating a toxic
transgene-containing pRCASBP vector into the limb bud,
we can overcome this difficulty, although we anticipate
a slow expansion of transgene expression in this case.
Combination of expression vectors and other useful vectors
Another advantage of electroporation includes the ability
to co-electroporate two or three expression plasmids
simultaneously. This can be used for the visualization
of an electroporated area, by mixing pCAGGS-EGFP
or pCMV-EGFP vectors in a plasmid cocktail and
subsequent expression of GFP fluorescent signals in
limb bud. Using our techniques, we can successfully
misexpress two or three transgenes in the limb bud in
a single electroporation experiment. This is a significant
advantage for us, since combinatorial actions of genes,
for example transcription factors, are observed in vivo.
In the RCASBP system, there are available subgroups A, B, D, and E (Logan & Tabin 1998). RCASBP
subgroup A, B, and D could be infected in C/O (cells
susceptible to all subgroups of avian leukosis viruses)
egg limbs (Suzuki et al. unpubl. data, 2008), so it is
possible to overexpress at least three transgenes at
the same time using the RCASBP system.
Protocols for electroporation into limb
Egg incubation and exposure
CUY21 electroporator (Nepa gene, Ichikawa, Japan),
dissecting microscope (Leica MZAPO, Wetzlar, Germany),
and platinum electrodes (CUY613P2, Nepa gene) are
set up (Fig. 2A).
Incubate eggs in a humidified incubator (IC800,
Yamato, Tokyo, Japan) at 39°C until St. 13/14, kept down
for easy access (Fig. 2B). One can also position eggs
in a top-down manner, but this positioning might not
provide enough of a window to inject plasmids and
an efficient configuration of electrodes on the surface of
the embryo. In addition, a flat aspect of the egg can
be patched much more easily than the top of the egg
using plastic tape. Complete sealant of windows is one
critical factor necessary to ensure high viability of the
embryos, preventing bacterial and/or fungal contamination after electroporation.
After incubating eggs until St. 13/14, an 18G1/2 needle
(NN-1838R, Terumo, Tokyo, Japan) or forceps (Dumont
No. 5 forceps, 11 cm; No. 500341, World precision
instruments) should be used to create a window at
the broader edge of the eggshell. Approximately 5 mL
of albumin should be gently removed from this hole
with a 25-mL syringe (SS-20ESZ, Terumo). Using
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this procedure, one can procure enough space inside
the egg for electroporation. This tiny hole can then
be immediately sealed with plastic tape (Fig. 2B). Subsequently, one can obtain access by cutting the eggshell with a (pair of) curved scissors. One should be sure
that this window is wide enough for electroporation,
while avoiding the creation of a window that is too
wide, approximately bigger than 4 cm2, which could
instead damage the embryo and/or lead to infection.
Through this window, one can observe the living embryo.
Preparation of tungsten needles and glass capillaries
Chick embryos develop between the yolk sac and
vitelline membrane. The membrane must be removed
to expose the surface of the embryo using a tungsten
needle, which has been sharpened precisely for this
undertaking. For sharpening, tungsten wire tips
0.20 mm in diameter (No. 461267, Nilaco Co., Tokyo,
Japan) are immersed in 1N NaOH. Electric current is
then applied at 23 V. This method yields sharp tips,
which can then be checked under a microscope.
Glass capillaries for DNA injection are made by pulling
Narishige glass capillaries (1 × 90 mm glass capillaries
with filament; GPC-1, Narishige, Tokyo, Japan) with
a Narishige puller (PC-10, Narishige), pre-adjusted for
the desired degree of sharpness. There are three kinds
of weights in the Narishige puller; two large weights
(93 g) and one smaller weight (24 g), which are used
to produce different pulled capillary tubes according
to varying specifications. We typically use one large
weight and one small weight. Using this combination,
we can make glass capillaries with a natural curve
after pulling. These curved capillaries are very useful
for injecting DNA solution into a thinner body wall. An
alternative means of producing curved capillaries involves
bending the tip of glass capillaries with no. 5 forceps
immediately after pulling. The curved needle tip is broken
off by a no. 5 forceps under a dissecting microscope,
and placed at the end of an aspirator tube.
DNA solution preparation
The pCAGGS or RCASBP vector is next diluted with
distilled water to a final concentration of 3–5 μg/μL
and mixed with pCAGGS-EGFP expression vector
(0.8 –1.0 μg/μL) for electroporation. This concentration
of the pCAGGS-EGFP vector is enough to visualize
the entire electroporated area (Momose et al. 1999).
Viscous DNA in solution (a concentration greater than
5 μg/μL) decreases the efficiency of electroporation and
the subsequent expression of the transgene of interest.
A cocktail of expression plasmids can then be
placed on the cap of a PCR tube (0.2 mL), and mixed
© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists
462
T. Suzuki and T. Ogura
Fig. 2. Electroporation into the limb field. (A) CUY21 electroporator, dissecting microscope, and platinum electrodes are set up. (B)
Anode electrode and cathode electrode are shown on the right and left, respectively. The egg is arranged lying down. (C) After injection
of Rotring-phosphate-buffered saline (PBS) solution, a St. 14 chick embryo is highlighted on the black background. (D) Forelimb and
hindlimb fields are shown by a yellow circle. (E) Expression pattern of cfgf10 at St. 14 in a horizontal section of the forelimb field. cfgf10
is expressed at the somatic lateral plate mesoderm (LPM) and nephric primordium. (F) A cathode electrode is inserted under the embryo.
(G) A cathode electrode is placed under the forelimb field. (H) The vitelline artery is labeled by India ink injection. (I) Fast Green-DNA
solution is injected into the forelimb field. (J) An anode electrode is prepared on the forelimb field. The anode electrode is placed in the
center of the forelimb field (K), anterior (L), posterior (M), or parallel to cathode electrode (N). (O) The hindlimb field is shown by a yellow
circle. (P) Fast Green-DNA solution is injected into the hindlimb field. (Q) An anode electrode is placed in the center of the hindlimb field.
with a solution of 1% Fast Green (diluted in PBS; No.
061-00031, Wako, Osaka, Japan) before electroporation. Coloring the DNA solution is important to
obtain high electroporation efficiency. However, a concentration of Fast Green that is too high, greater than
1%, might decrease the efficiency of transfection. This
electroporation cocktail is then vacuum-extracted by
a curved glass capillary attached to an aspirator tube.
Injection of DNA solution into limb fields
After opening an eggshell, one can observe the chick
embryo under the vitelline membrane. At this moment,
a small amount of a Rotring-phosphate-buffered
saline (PBS) solution (PBS with 1:40 Rotring; Art-R
591017, Sanford Co., Tokyo, Japan) (approximately
100–200 μL) is injected from outside of the sinus terminalis located near the tail bud, using a syringe
equipped with a 26G1/2 needle (NN-2613S, Terumo).
This produces a black background, which provides a
higher degree of contrast with which to visualize the
embryo (Fig. 2C). After bathing the embryo in 1 mL of
sterilized PBS, vitelline membrane near either the forelimb or hindlimb field is gently shorn by a sharpened
tungsten needle (Fig. 2D). At St. 14 (the best stage
for electroporation into limb buds) 22 pairs of somites
© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists
Electroporation into chick limb buds
are present. DNA solution is injected into an embryonic
space located between the somatic LPM and the
splanchnic LPM (Fig. 2E). An L-shaped platinum
cathode is inserted from the hole that was created
for injection of the Rotring-PBS solution (Fig. 2F), and
placed under either the forelimb (Fig. 2G) or hindlimb
field (Fig. 2O). As described above, the forelimb field
forms at the 15–20 somite level of the LPM, spanning
from an extended edge of the anterior LPM to a
location corresponding to the position of the vitelline
artery (Fig. 2H). At this stage, it is not easy to locate
the hindlimb field by counting somites. Instead, one
can gauge its position by checking the expression
domain of cfgf10, which spans approximately six
somite lengths from the posterior edge of the
LPM, in the boundary between the body wall and
amnion.
To inject DNA solution into the forelimb field, the
tip of a curved glass capillary tube is inserted from
the anterior side of the forelimb field by pricking the
thin embryonic tissue. For injection into the hindlimb
field, a glass capillary tip is inserted from the posterior
side of the hindlimb field. Care is required to avoid
any damage to the vitelline artery, which typically
results in the death of an embryo. After insertion of the
needle, DNA solution can be injected. After successful
injection, one can observe green pigment in the
Fast Green-filling forelimb (Fig. 2I) or hindlimb field
(Fig. 2P).
Electroporation into limb fields
We developed three different methods by modifying
our original protocol (Ogura 2002), typically using a
platinum anode (Fig. 2J).
1. When an anode is placed at the center of either
the forelimb (Fig. 2K) or the hindlimb field (Fig. 2Q)
before electric pulses (8 V, 60 ms pulse-on, 50 ms
pulse-off, three repetitions), one can express
transgene along the entire limb bud, with strong
expression along the middle part of the limb. By
positioning an anode at either the anterior side
(Fig. 2L) or the posterior side of the limb field
(Fig. 2M),
transgene
expression
becomes
restricted to the anterior or posterior portions,
respectively.
2. One can expand the domain of transgene expression
by moving an anode serially. In this case, the
electroporator is set for three pulses (8 V, 60 ms
pulse-on, 1 second pulse-off, for three repetitions).
For the first pulse, an anode is placed in the
anterior limb field (Fig. 2L). During a 1 second
pulse-off pause, this anode is moved to the central
part of the limb field (Fig. 2K), and then a second
463
pulse is applied. The anode is moved further to the
posterior limb field (Fig. 2M) for the last electric
pulse. By applying three electric pulses serially in
different parts of the limb field, one can overexpress
transgene strongly and uniformly in the whole limb
bud.
3. An anode can be placed over the limb field, making
a parallel configuration with a cathode (Fig. 2N) for
electric pulses (5 V, 60 ms pulse-on, 50 ms pulse-off,
three repetitions). In this setting, one can misexpress transgene throughout the entire limb bud.
However, lower voltage pulses must be used, since
the electric field formed between two parallel
electrodes is wider and a higher voltage in this
setting may damage cells.
During electroporation, two electrodes must be kept
in solution, not touching the surface of the embryos
or the vitelline artery in order to avoid tissue damage.
These methods are efficient at somatic LPM from St.
12 to St. 16. At later stages, from St. 17, it is difficult
to keep DNA solution inside of the limb bud. One
may be able to electroporate at later stages using
these methods, but expression of the electroporated
plasmid would be weaker. When truncation or shortening of the limb buds is observed, even after electroporation with non-toxic pCAGGS-EGFP, this finding
must be replicated in order to confirm the electrodes
were free from the embryonic tissues, and not affecting
the experimental results.
After electroporation, the cathode is gently withdrawn
from the amnion, and 30 μL of a penicillin-streptomycin
solution (PBS with 1:100 penicillin-streptomycin; No.
15140-122, Invitrogen, Carlsbad, CA) (for 1 L 1 x PBS:
NaCl 5.8 g, NaH2PO42H2O 0.36 g, Na2HPO412H2O 2.76 g)
is used to bathe the embryo. The eggshell window
is then sealed firmly with plastic tape. Embryos
should be incubated again immediately after sealing.
When pCAGGS-EGFP (5 μg/μL) is electroporated
into the forelimb field, EGFP expression can be detected
after 1–3 h (Fig. 3A–D). EGFP expression becomes
stronger at 6–12 h after electroporation (Fig. 3E–H),
and expands until 24–48 h (Fig. 3I–L), probably due
to proliferation and migration of cells. When a lower
concentration of pCAGGS-EGFP (0.1 μg/μL) is used,
weak expression of EGFP can be detected at 12 h,
whereas electroporation of 1 μg/μL of pCAGGS-EGFP
results in an intermediate level of expression (Fig. 3M–P).
Robust EGFP expression can be observed in the
hindlimb field at 12 h after electroporation of 5 μg/μL
pCAGGS-EGFP (Fig. 3Q,R). By changing the positions
of an anode as shown in Figure 2K–M, one can target
expression of the transgene of interest to either the
anterior (Fig. 3S), middle (Fig. 3T) or posterior side
(Fig. 3U) of the limb bud.
© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists
464
T. Suzuki and T. Ogura
Fig. 3. Expression pattern of enhanced green fluorescent protein (EGFP) after electroporation into the limb field. (A–L) Time course of
EGFP expression after 5 μg/μL of pCAGGS-EGFP was electroporated into the forelimb field. EGFP expression is detected at 3 h after
electroporation (yellow arrow in C). Lower panels show high magnification of upper panels, respectively. 0.1 μg/μL or 1.0 μg/μL of
pCAGGS-EGFP was electroporated into the forelimb field, and EGFP expression was detected 12 h after electroporation (M and N, O and
P). (Q and R) 5 μg/μL of pCAGGS-EGFP was electroporated into the hindlimb. 5 μg/μL of pCAGGS-EGFP was electroporated into the anterior
limb field (S), middle limb field (T), or posterior limb field (U), respectively, and EGFP fluorescence was detected 24 h after electroporation.
Trouble shooting
Q: We cannot detect any EGFP expression.
A: First, be sure the plasmid expresses in other systems,
such as in cultured cells. Is the concentration of the
plasmid correct and adjusted to a range of 3–5 μg/μL,
or is it potentially too high?
We observe bubbles around the electrodes after
electroporation. If you did not observe bubbles, you
should move your electrode closer to the body of the
embryo, but do not touch the embryonic tissues directly.
Also, be sure that you inject enough DNA into the
embryonic column. Successful injection can be judged
by visualization of the Fast Green-DNA cocktail in the
embryonic tissues. After the DNA solution is injected,
electroporate as quickly as possible. Otherwise, DNA
will diffuse out of the electroporation field before
sufficient plasmid can be taken up by the cells.
Q: We could not get EGFP expression beneath the
AER.
A: Attempt to move the electrodes more laterally.
Q: We got a truncated or a shorter limb bud when we
electroporated with pCAGGS-EGFP.
A: Adjust the distance between electrodes and the voltage of the electric pulses you use. By lowering the
voltage, such artifacts will often be prevented. Voltages
below 5 V are not typically effective.
Q: We need stronger expression.
© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists
Electroporation into chick limb buds
A: Move the electrodes closer to the embryos and/or
increase the electroporation voltage. If the concentration
of plasmid is less than 5 μg/μL, concentrate it. Since the
pCAGGS vector shows the most effective transgene
transcription in the limb, use this vector.
Q: Malformations developed outside of the limb.
A: Your electrodes are likely too close to the midline
tissue of the embryo. Move your electrodes to a more
lateral position. Arrange two electrodes closer together to
create a narrower pulse field and apply lower voltages.
Q: Our embryos do not survive well after electroporation.
A: It is best to check viability of the embryos 24 h after
electroporation. If you see malformation even at this
early time point, your electroporation setting is probably
too strong. Check your electroporation conditions using
the points described above. If no malformation develops, there might instead be a problem(s) with the egg,
incubation or sealant of the eggshell. Make sure that
you use fresh eggs, re-incubate them immediately after
electroporation and seal the eggshell window completely.
Acknowledgments
Special thanks to Minoru Omi for helpful discussions.
This work was funded by Japan Society for the Promotion
of Science to T.S., and Takeda Science Foundation to
T.O.
465
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© 2008 The Authors
Journal compilation © 2008 Japanese Society of Developmental Biologists