209
Advances in Genome Editing Tools
it requires the use of restriction enzyme-based or Gibson
Assembly cloning to introduce the promoters of interest
(Das and Brown, 2004a ; Kerney et al., 2012; Rankin et al.,
2011). Alternatively, another destination vector, pDXTP, can
be used in conjunction with pDXTR. pDXTP is compatible
with the promoter entry vectors included in pTransgenesis,
allowing rapid recombination of the promoter upstream of
the doxycycline inducible transcription factor rtTA, thus
maintaining the ability to use the Tet-On system to control
transgene expression. One shortcoming of the pDXTR and
pDXTP plasmids is that they are relatively large, and both
contain several insulator sequences. This makes pDXTR
particularly prone to self-recombination following transformation into E. coli, which can be prevented by growing the
transformed bacteria at a lower temperature.
The ability to regulate transgene activity in both time and
space expands the breadth of experimental questions that
can be investigated using transgenics in Xenopus. A number
of cell- and tissue-specifc promoters have been characterized and used for spatial regulation of transgene activity in
Xenopus (Horb et al., 2019). In addition to the Tet-On system, temporal control of transgene expression in Xenopus
has been achieved using the temperature-inducible hsp70
promoter or the modifed dual-component GAL4-UAS system with the GAL4 fused to the ligand binding domain of
the progesterone receptor (PR) (Beck et al., 2006; Horb et
al., 2019). Finally, transgenic lines expressing Cre have been
used as drivers to induce switches in f uorescence following
a cross to lines with loxP sites (Roose et al., 2009; Waldner
et al., 2006). These diverse methods of transgenesis, transgenic vector construction, and transgene activity regulation
provide a solid framework within which Xenopus researchers can design experiments to study biological processes
through genome modif cation.
14.2. USES OF TRANSGENIC XENOPUS
The way transgenesis would expand the experimental toolkit in Xenopus was made evident with the f rst transgenesis paper by Kroll and Amaya, in which they examined the
temporal requirement of FGF signaling in early Xenopus
development. Previous experiments suggested that FGF
signaling was necessary for primary mesoderm induction
as well for later processes, including maintenance of mesoderm fate and neural induction and patterning. Mesoderm
induction occurs quite early in embryogenesis and can be
studied using mRNA injections, but later events require
FGF signaling to be perturbed after mesoderm induction.
Transgenesis was used to express a dominant negative
FGF receptor after mesoderm induction and showed that
while FGF was required for maintenance of mesoderm
fate, it was not required for neural induction and patterning (Kroll and Amaya, 1996 ). Its lack of involvement in
neural induction contradicted previous data, revealing
how transgenics could improve data quality in the frog.
In addition to dominant negative protein expression, other
loss-of-function approaches, such as shRNA, have also
proven effective in Xenopus transgenics (Edholm and
Robert, 2018).
Using inducible or tissue-specifc promoters allows
research on developmental processes that occur several days
to months after fertilization, including regeneration and
metamorphosis. Regeneration is thought to occur through
the reactivation of the same program involved in normal
development, but this cannot be studied in traditional knockout experiments, since the target tissue/organ may be perturbed by loss of function early in development. In Xenopus,
tadpole tails regenerate upon amputation from three days
after fertilization until metamorphosis, except during a short
refractory period at four to fve days. Using a heat shockinducible hsp70 promoter, Beck et al. (2003) showed that
reactivation of the BMP and Notch signaling pathways during this refractory period promoted regeneration, whereas
their inhibition at other stages blocked regeneration. In F0
transgenics, they found variability in this ability, possibly
due to integration site and transgene expression levels, but
in F1 transgenics, the phenotype was more consistent. These
results suggested that generating stable transgenic lines produces more robust results.
Metamorphosis in Xenopus is a model for human perinatal endocrinology when multiple hormones regulate
many aspects of tissue growth, development, remodeling, and maturation (Buchholz, 2015). This late-stage
event occurs 30–60 days after fertilization, and transgenesis in Xenopus was essential for in vivo functional studies (Marsh-Armstrong et al., 2004; Mukhi et al., 2008,
2009; Schreiber et al., 2001). In particular, binary-inducible
transgenic systems have proven useful for temporal and
tissue-specifc control of transgenes during metamorphosis
( Buchholz, 2012 ; Das and Brown, 2004b ). The tetracycline
(Tet)-inducible system allows for tight control of transgene
expression by simple addition of doxycycline (Dox) to the
water. This system requires two different transgenes: one
promoter (tissue-specifc or ubiquitous) to control expression of rtTA (a Dox-dependent transcription factor) and a
second tetracycline-inducible (TRE) promoter to control
expression of the gene of interest. This system elucidated
several aspects of metamorphosis, including gene switching,
transdifferentiation of pancreatic acinar cells to ductal cells,
and limb development (Brown et al., 2005; Cai et al., 2007;
Mukhi and Brown, 2011; Mukhi et al., 2010).
Another beneft of Xenopus transgenics is the ability to
use promoters from other species, including rat, mouse,
and zebrafsh, to drive expression in a tissue-specif c manner (Beck and Slack, 1999; Love et al., 2011a). This can
be used to study the ability of factors to convert one tissue
to another. Combining such expression with a secondary
reporter to label the tissue generated allows monitoring of
transdifferentiation events in real time. For example, using
a murine transthyretin promoter to drive expression of pancreatic transcription factors in the liver, combined with the
rat elastase promoter driving GFP, it was found that only two
pancreatic transcription factors, Ptf1a and Pdx1, were able
to convert liver to pancreas (Horb et al., 2003; Jarikji et al.,
Advances in Genome Editing Tools
it requires the use of restriction enzyme-based or Gibson
Assembly cloning to introduce the promoters of interest
(Das and Brown, 2004a ; Kerney et al., 2012; Rankin et al.,
2011). Alternatively, another destination vector, pDXTP, can
be used in conjunction with pDXTR. pDXTP is compatible
with the promoter entry vectors included in pTransgenesis,
allowing rapid recombination of the promoter upstream of
the doxycycline inducible transcription factor rtTA, thus
maintaining the ability to use the Tet-On system to control
transgene expression. One shortcoming of the pDXTR and
pDXTP plasmids is that they are relatively large, and both
contain several insulator sequences. This makes pDXTR
particularly prone to self-recombination following transformation into E. coli, which can be prevented by growing the
transformed bacteria at a lower temperature.
The ability to regulate transgene activity in both time and
space expands the breadth of experimental questions that
can be investigated using transgenics in Xenopus. A number
of cell- and tissue-specifc promoters have been characterized and used for spatial regulation of transgene activity in
Xenopus (Horb et al., 2019). In addition to the Tet-On system, temporal control of transgene expression in Xenopus
has been achieved using the temperature-inducible hsp70
promoter or the modifed dual-component GAL4-UAS system with the GAL4 fused to the ligand binding domain of
the progesterone receptor (PR) (Beck et al., 2006; Horb et
al., 2019). Finally, transgenic lines expressing Cre have been
used as drivers to induce switches in f uorescence following
a cross to lines with loxP sites (Roose et al., 2009; Waldner
et al., 2006). These diverse methods of transgenesis, transgenic vector construction, and transgene activity regulation
provide a solid framework within which Xenopus researchers can design experiments to study biological processes
through genome modif cation.
14.2. USES OF TRANSGENIC XENOPUS
The way transgenesis would expand the experimental toolkit in Xenopus was made evident with the f rst transgenesis paper by Kroll and Amaya, in which they examined the
temporal requirement of FGF signaling in early Xenopus
development. Previous experiments suggested that FGF
signaling was necessary for primary mesoderm induction
as well for later processes, including maintenance of mesoderm fate and neural induction and patterning. Mesoderm
induction occurs quite early in embryogenesis and can be
studied using mRNA injections, but later events require
FGF signaling to be perturbed after mesoderm induction.
Transgenesis was used to express a dominant negative
FGF receptor after mesoderm induction and showed that
while FGF was required for maintenance of mesoderm
fate, it was not required for neural induction and patterning (Kroll and Amaya, 1996 ). Its lack of involvement in
neural induction contradicted previous data, revealing
how transgenics could improve data quality in the frog.
In addition to dominant negative protein expression, other
loss-of-function approaches, such as shRNA, have also
proven effective in Xenopus transgenics (Edholm and
Robert, 2018).
Using inducible or tissue-specifc promoters allows
research on developmental processes that occur several days
to months after fertilization, including regeneration and
metamorphosis. Regeneration is thought to occur through
the reactivation of the same program involved in normal
development, but this cannot be studied in traditional knockout experiments, since the target tissue/organ may be perturbed by loss of function early in development. In Xenopus,
tadpole tails regenerate upon amputation from three days
after fertilization until metamorphosis, except during a short
refractory period at four to fve days. Using a heat shockinducible hsp70 promoter, Beck et al. (2003) showed that
reactivation of the BMP and Notch signaling pathways during this refractory period promoted regeneration, whereas
their inhibition at other stages blocked regeneration. In F0
transgenics, they found variability in this ability, possibly
due to integration site and transgene expression levels, but
in F1 transgenics, the phenotype was more consistent. These
results suggested that generating stable transgenic lines produces more robust results.
Metamorphosis in Xenopus is a model for human perinatal endocrinology when multiple hormones regulate
many aspects of tissue growth, development, remodeling, and maturation (Buchholz, 2015). This late-stage
event occurs 30–60 days after fertilization, and transgenesis in Xenopus was essential for in vivo functional studies (Marsh-Armstrong et al., 2004; Mukhi et al., 2008,
2009; Schreiber et al., 2001). In particular, binary-inducible
transgenic systems have proven useful for temporal and
tissue-specifc control of transgenes during metamorphosis
( Buchholz, 2012 ; Das and Brown, 2004b ). The tetracycline
(Tet)-inducible system allows for tight control of transgene
expression by simple addition of doxycycline (Dox) to the
water. This system requires two different transgenes: one
promoter (tissue-specifc or ubiquitous) to control expression of rtTA (a Dox-dependent transcription factor) and a
second tetracycline-inducible (TRE) promoter to control
expression of the gene of interest. This system elucidated
several aspects of metamorphosis, including gene switching,
transdifferentiation of pancreatic acinar cells to ductal cells,
and limb development (Brown et al., 2005; Cai et al., 2007;
Mukhi and Brown, 2011; Mukhi et al., 2010).
Another beneft of Xenopus transgenics is the ability to
use promoters from other species, including rat, mouse,
and zebrafsh, to drive expression in a tissue-specif c manner (Beck and Slack, 1999; Love et al., 2011a). This can
be used to study the ability of factors to convert one tissue
to another. Combining such expression with a secondary
reporter to label the tissue generated allows monitoring of
transdifferentiation events in real time. For example, using
a murine transthyretin promoter to drive expression of pancreatic transcription factors in the liver, combined with the
rat elastase promoter driving GFP, it was found that only two
pancreatic transcription factors, Ptf1a and Pdx1, were able
to convert liver to pancreas (Horb et al., 2003; Jarikji et al.,