208
Xenopus
thousands of eggs at a time, and of those normal survivors,
as many as 36% show stable, non-mosaic transgene expression. Furthermore, F0 animals that show strong non-mosaic
expression almost invariably transmit the transgene through
their germline (Marsh-Armstrong et al., 1999). This was a
major improvement in transgenic effciency over the results
obtained by injection of linearized plasmid DNA into fertilized embryos and also works effciently in Xenopus tropicalis ( Offeld et al., 2000). The major limitation of REMI is
that it is highly mutagenic, capable of generating up to four
different transgene integration sites with multiple integrations in the injected individual, complicating interpretation
of phenotypic analyses (Bronchain et al., 1999; MarshArmstrong et al., 1999). Furthermore, studies analyzing late
developmental stages are not trivial in the F0 generation due
to the small number of survivors (Chesneau et al., 2008).
In 2000, REMI was simplifed by eliminating the need for
restriction enzyme and egg extract while retaining similar
effciency (Sparrow et al., 2000).
Following REMI, the successful innovations in transgenesis techniques focused on increasing rates of survival
and normal development, mainly through injection of transgenic reagents into embryos rather than eggs, resulting in
less technically demanding procedures and greater normal development. First, a relatively simple method relying
on co-injection of the commercially available rare-cutting
meganuclease, I-SceI, and transgenic DNA carrying the 18
base-pair long I-SceI recognition site was effcient in both
Xenopus laevis and Xenopus tropicalis (Pan et al., 2006).
The observed survival rate past metamorphosis was greater
than 55%, with 10% to 12% of the survivors showing nonmosaic transgene expression (Pan et al., 2006). Furthermore,
only one to eight copies of the transgene integrated at up to
two distinct integration sites.
A second similarly simple technique involves microinjection of mRNA encoding the φC31 bacteriophage integrase
with a transgenic plasmid DNA containing a 34 base-pair
long bacterial attachment site, attB (Allen and Weeks,
2005; Li et al., 2012). The integrase mediates recombination between the attB site and a 39 base-pair long phagedependent attachment site attP; recombination, however,
can also occur at pseudo-attP sites with similarity as low
as 24% to the phage attP sequence, though at much lower
effciencies (Groth et al., 2004; Thyagarajan et al., 2001).
The main advantage of this approach is that it is thought to
result in integration of a single transgene into the Xenopus
genome, although reports of successful germline transmission are lacking (Allen and Weeks, 2005; Li et al., 2012).
The European Xenopus Resource Centre (EXRC) has an
engineered transgenic line that contains an attP docking
site within a functional cyan fuorescent protein coding
sequence. The use of this line in conjunction with φC31 integrase may provide a highly effcient way to generate novel
transgenics, screened by loss of cyan f uorescence (Horb
et al., 2019).
Finally, two approaches based on the use of transposasedriven transgene integration, using Sleeping Beauty or Tol2,
have been effective in Xenopus. Both involve co-injection of
the transposase, either as mRNA or protein, with the transposon or DNA encoding the transgenic package f anked by
transposase target sequences (Hamlet et al., 2006; Shibano
et al., 2007; Sinzelle et al., 2006; Yergeau et al., 2009).
Uniquely, this method can also be used in a variant of gene
and enhancer trap, in which forward genetic experiments
are achieved by secondarily remobilizing the transgene following reintroduction of the transposase ( Lane et al., 2013;
Yergeau et al., 2011a, 2012).
14.1.3. PTRANSGENESIS: STREAMLINING
TRANSGENE CONSTRUCTION
Besides novel ways of promoting transgene integration into
the Xenopus genome, a major innovation came through use
of modular Gateway cloning to streamline the generation of
transgenic plasmids. The pTransgenesis system uses multisite Gateway technology to allow for rapid generation of
transgenic plasmids via recombination of a destination vector
containing DNA sequences required for genome integration
and three entry clones, a fuorescent transgenesis reporter, a
promoter, and a coding sequence (Love et al., 2011b). The
destination vectors allow a choice among transgenic methods, including I-SceI meganuclease, Tol2 transposase, and
φC31 integrase. Novel entry clones can be easily generated
through simple recombination of a PCR product and donor
vector, and existing entry clones can be mixed and matched
to generate a diverse range of transgenic plasmids, making
this a very powerful and f exible system.
There are, however, a number of issues with this system.
First, the particular donor vectors used to generate pTransgenesis entry clones stopped being commercially available
shortly after publication. Nonetheless, they can be procured
from the Zebrafsh International Resource Center (ZIRC)
as part of the Tol2kit used for transgenic plasmid creation
in zebrafsh (Kwan et al., 2007). Second, only three of the
four destination vectors contain chicken beta-globin HS4
insulator sequences that have been shown to reduce integration site effects on transgene expression (Allen and Weeks,
2005). Third, one of the vectors includes two I-SceI target
sequences fanking the transgene, which may increase the
effciency of transgenesis but risks integration of the vector
backbone independent of the transgene sequence. Fourth, the
pTransgenesis system, as constructed, is not compatible with
the Xenopus ORFeome (Grant et al., 2015). The recombination sites used in the ORFeome constructs are the same ones
as those used in the pTransgenesis promoter entry plasmids
and combining both systems misplaces the ORF within the
transgenic construct. Instead, a newer two-plasmid system
can be used in conjunction with the ORFeome to rapidly
generate transgenic plasmids (Sterner et al., 2019). Like
pTransgenesis, this system is versatile and permits selection
of the transgenic technique: I-SceI, Tol2, and φC31. One of
the destination vectors, pDXTR, allows for rapid gateway
recombination with the ORFeome plasmids and includes the
Tet-On system for inducible transgene expression. However,
Xenopus
thousands of eggs at a time, and of those normal survivors,
as many as 36% show stable, non-mosaic transgene expression. Furthermore, F0 animals that show strong non-mosaic
expression almost invariably transmit the transgene through
their germline (Marsh-Armstrong et al., 1999). This was a
major improvement in transgenic effciency over the results
obtained by injection of linearized plasmid DNA into fertilized embryos and also works effciently in Xenopus tropicalis ( Offeld et al., 2000). The major limitation of REMI is
that it is highly mutagenic, capable of generating up to four
different transgene integration sites with multiple integrations in the injected individual, complicating interpretation
of phenotypic analyses (Bronchain et al., 1999; MarshArmstrong et al., 1999). Furthermore, studies analyzing late
developmental stages are not trivial in the F0 generation due
to the small number of survivors (Chesneau et al., 2008).
In 2000, REMI was simplifed by eliminating the need for
restriction enzyme and egg extract while retaining similar
effciency (Sparrow et al., 2000).
Following REMI, the successful innovations in transgenesis techniques focused on increasing rates of survival
and normal development, mainly through injection of transgenic reagents into embryos rather than eggs, resulting in
less technically demanding procedures and greater normal development. First, a relatively simple method relying
on co-injection of the commercially available rare-cutting
meganuclease, I-SceI, and transgenic DNA carrying the 18
base-pair long I-SceI recognition site was effcient in both
Xenopus laevis and Xenopus tropicalis (Pan et al., 2006).
The observed survival rate past metamorphosis was greater
than 55%, with 10% to 12% of the survivors showing nonmosaic transgene expression (Pan et al., 2006). Furthermore,
only one to eight copies of the transgene integrated at up to
two distinct integration sites.
A second similarly simple technique involves microinjection of mRNA encoding the φC31 bacteriophage integrase
with a transgenic plasmid DNA containing a 34 base-pair
long bacterial attachment site, attB (Allen and Weeks,
2005; Li et al., 2012). The integrase mediates recombination between the attB site and a 39 base-pair long phagedependent attachment site attP; recombination, however,
can also occur at pseudo-attP sites with similarity as low
as 24% to the phage attP sequence, though at much lower
effciencies (Groth et al., 2004; Thyagarajan et al., 2001).
The main advantage of this approach is that it is thought to
result in integration of a single transgene into the Xenopus
genome, although reports of successful germline transmission are lacking (Allen and Weeks, 2005; Li et al., 2012).
The European Xenopus Resource Centre (EXRC) has an
engineered transgenic line that contains an attP docking
site within a functional cyan fuorescent protein coding
sequence. The use of this line in conjunction with φC31 integrase may provide a highly effcient way to generate novel
transgenics, screened by loss of cyan f uorescence (Horb
et al., 2019).
Finally, two approaches based on the use of transposasedriven transgene integration, using Sleeping Beauty or Tol2,
have been effective in Xenopus. Both involve co-injection of
the transposase, either as mRNA or protein, with the transposon or DNA encoding the transgenic package f anked by
transposase target sequences (Hamlet et al., 2006; Shibano
et al., 2007; Sinzelle et al., 2006; Yergeau et al., 2009).
Uniquely, this method can also be used in a variant of gene
and enhancer trap, in which forward genetic experiments
are achieved by secondarily remobilizing the transgene following reintroduction of the transposase ( Lane et al., 2013;
Yergeau et al., 2011a, 2012).
14.1.3. PTRANSGENESIS: STREAMLINING
TRANSGENE CONSTRUCTION
Besides novel ways of promoting transgene integration into
the Xenopus genome, a major innovation came through use
of modular Gateway cloning to streamline the generation of
transgenic plasmids. The pTransgenesis system uses multisite Gateway technology to allow for rapid generation of
transgenic plasmids via recombination of a destination vector
containing DNA sequences required for genome integration
and three entry clones, a fuorescent transgenesis reporter, a
promoter, and a coding sequence (Love et al., 2011b). The
destination vectors allow a choice among transgenic methods, including I-SceI meganuclease, Tol2 transposase, and
φC31 integrase. Novel entry clones can be easily generated
through simple recombination of a PCR product and donor
vector, and existing entry clones can be mixed and matched
to generate a diverse range of transgenic plasmids, making
this a very powerful and f exible system.
There are, however, a number of issues with this system.
First, the particular donor vectors used to generate pTransgenesis entry clones stopped being commercially available
shortly after publication. Nonetheless, they can be procured
from the Zebrafsh International Resource Center (ZIRC)
as part of the Tol2kit used for transgenic plasmid creation
in zebrafsh (Kwan et al., 2007). Second, only three of the
four destination vectors contain chicken beta-globin HS4
insulator sequences that have been shown to reduce integration site effects on transgene expression (Allen and Weeks,
2005). Third, one of the vectors includes two I-SceI target
sequences fanking the transgene, which may increase the
effciency of transgenesis but risks integration of the vector
backbone independent of the transgene sequence. Fourth, the
pTransgenesis system, as constructed, is not compatible with
the Xenopus ORFeome (Grant et al., 2015). The recombination sites used in the ORFeome constructs are the same ones
as those used in the pTransgenesis promoter entry plasmids
and combining both systems misplaces the ORF within the
transgenic construct. Instead, a newer two-plasmid system
can be used in conjunction with the ORFeome to rapidly
generate transgenic plasmids (Sterner et al., 2019). Like
pTransgenesis, this system is versatile and permits selection
of the transgenic technique: I-SceI, Tol2, and φC31. One of
the destination vectors, pDXTR, allows for rapid gateway
recombination with the ORFeome plasmids and includes the
Tet-On system for inducible transgene expression. However,