316
Xenopus
22.3. PRESENT STATE OF THE FIELD
22.3.1. THE ADVANTAGES OF OUTBRED LINES
Another complication with inbred lines is the different phenotypes observed for the same mutation on different genetic
backgrounds (Montagutelli, 2000). For example, deletion
of the EGF receptor is peri-implantation lethal on a CF-1
background, but in CD-1 mice, the pups live for up to three
weeks postnatally (Threadgill et al., 1995). Differences in
the severity of phenotypes linked to which inbred line was
used have been reported for gene mutations involved in polycystic kidney disease, tooth development, spermatogenesis,
and craniofacial development ( Li et al., 2013; Percival et al.,
2017; Sakai et al., 2019; Sommardahl et al., 2001). Likewise,
different lines show different susceptibility to neural tube
defects in mice (Leduc et al., 2017), and to prenatal alcohol exposure, resulting in FASD in mice, rats, chickens, and
zebrafsh (Eberhart and Parnell, 2016).
For several decades, our reasoning has focused on the
benefts of using inbred lines to minimize the inf uence
of polymorphisms when examining single gene function.
Now we recognize that there are many advantages to using
outbred models. First and foremost, the lack of genetic
diversity in inbred strains may not adequately inform studies in patient populations, which are genetically diverse
(Justice and Dhillon, 2016). Recently, there has been a call
for including biological variability in animal-based biomedical research, including the use of genetic polymorphisms
(Voelkl et al., 2020). In support of using genetically heterogeneous animal models, it was recently shown that for the
majority of phenotypes assessed, outbred mice were as phenotypically stable as inbred mice and were more resistant
to housing differences between laboratories (Tuttle et al.,
2018). The advantages of outbred strains were acknowledged by developing genetically diverse strains, including
the Collaborative Cross mice, the Diversity Outbred mice
(Harrill and McAllister, 2017; Saul et al., 2019; Threadgill
et al., 2011), and the T5D zebrafsh line ( Balik-Meisner et al.,
2018 ).
22.3.2. USING XENOPUS TO STUDY THE IMPACT OF GENETIC
VARIATION ON DEVELOPMENT AND DISEASE
22.3.2.1. Most Xenopus Laboratory Populations
Are Genetically Diverse
Genetically diverse, outbred Xenopus populations have
been the traditional source of a rich history of biomedical
research. Their genetic diversity derives from three primary factors: outbred commercial stocks, random matings
to obtain experimental offspring, and design strategies to
increase diversity within an experimental group.
Most laboratories using Xenopus do not establish their
breeding colony by growing up the embryos produced
by the colony. Instead, they house colonies of up to several hundred sexually mature individuals purchased from
commercial frog breeders, such as Xenopus 1, Xenopus
Express, and Nasco. These commercial suppliers maintain
large populations of outbred adults that are sold to research
laboratories. Xenopus 1 maintains both a wild-caught and
a closed laboratory-bred colony, which are housed in separate facilities. For their laboratory-bred population, which
numbers approximately 20,000 including tadpoles, they
introduce about 1000 embryos from the wild-caught colony
every two to three years. These embryos are grown and bred
into the existing laboratory-bred colony to maintain genetic
diversity. Their wild-caught colony is a steady-state population of about 4500 adults replenished with f ve imports
from Chile per year; Xenopus were apparently introduced
into Chile in the 1950s (R. Weymouth, Xenopus 1, personal communication). The Nasco laboratory-bred colony
consisted of about 30,000 adults that were derived from
wild-caught founding adults imported from South Africa
between the early 1970s and 1996 (D. Brattlie, Nasco, personal communication). Although the Xenopus 1 and Nasco
populations are “closed,” their enormous sizes minimize
inbreeding and maximize genetic diversity. In both facilities, mating is random, producing laboratory-bred progeny
that are sold to research laboratories as well as added to the
breeding colony when they mature. Wild-caught animals
can be purchased from Xenopus 1 and Xenopus Express.
These animals are particularly favored by the research
community that uses oocytes for expression studies and
electrophysiology. Wild-caught animals are considered
more robust and disease resistant due to growing up in a
natural environment and are quarantined and tested for
pathogens before sale.
Typically, an individual laboratory maintains its breeding
colony of commercial and/or stock center acquired-adults to
produce offspring for experiments. Because the adults reproduce for up to 15 years, the colony within a single laboratory
often is composed of frogs that were purchased over many
years from more than one source. Accordingly, the adult
frogs in a given laboratory breeding colony are expected to
be unrelated and to display a high degree of genetic diversity, as shown by extensive EST and cDNA sequencing
(Blackshear et al., 2001; Fierro et al., 2007; Gilchrist et al.,
2004; Gilchrist and Pollet, 2012; Hellsten et al., 2007; Klein
et al., 2006). Even the comparison of RNAseq and genome
data between the inbred J and B strains revealed a degree of
sequence variation that resembled the situation determined
from comparing human genomes (Savova et al., 2017). Since
the typical commercial and laboratory breeding schemes
maintain a high level of genetic diversity in the offspring,
Xenopus has been proposed to be an ideal model to study
the functional relevance of specifc gene variants (Savova
et al., 2017).
The most common practices for generating offspring also
add genetic diversity to individual experiments. Typically,
embryos are produced by either natural mating of randomly
selected male and female pairs or by artif cial insemination
using the sperm of one randomly selected male to fertilize eggs from several randomly selected females. Because
embryos from different clutches might respond differently
Xenopus
22.3. PRESENT STATE OF THE FIELD
22.3.1. THE ADVANTAGES OF OUTBRED LINES
Another complication with inbred lines is the different phenotypes observed for the same mutation on different genetic
backgrounds (Montagutelli, 2000). For example, deletion
of the EGF receptor is peri-implantation lethal on a CF-1
background, but in CD-1 mice, the pups live for up to three
weeks postnatally (Threadgill et al., 1995). Differences in
the severity of phenotypes linked to which inbred line was
used have been reported for gene mutations involved in polycystic kidney disease, tooth development, spermatogenesis,
and craniofacial development ( Li et al., 2013; Percival et al.,
2017; Sakai et al., 2019; Sommardahl et al., 2001). Likewise,
different lines show different susceptibility to neural tube
defects in mice (Leduc et al., 2017), and to prenatal alcohol exposure, resulting in FASD in mice, rats, chickens, and
zebrafsh (Eberhart and Parnell, 2016).
For several decades, our reasoning has focused on the
benefts of using inbred lines to minimize the inf uence
of polymorphisms when examining single gene function.
Now we recognize that there are many advantages to using
outbred models. First and foremost, the lack of genetic
diversity in inbred strains may not adequately inform studies in patient populations, which are genetically diverse
(Justice and Dhillon, 2016). Recently, there has been a call
for including biological variability in animal-based biomedical research, including the use of genetic polymorphisms
(Voelkl et al., 2020). In support of using genetically heterogeneous animal models, it was recently shown that for the
majority of phenotypes assessed, outbred mice were as phenotypically stable as inbred mice and were more resistant
to housing differences between laboratories (Tuttle et al.,
2018). The advantages of outbred strains were acknowledged by developing genetically diverse strains, including
the Collaborative Cross mice, the Diversity Outbred mice
(Harrill and McAllister, 2017; Saul et al., 2019; Threadgill
et al., 2011), and the T5D zebrafsh line ( Balik-Meisner et al.,
2018 ).
22.3.2. USING XENOPUS TO STUDY THE IMPACT OF GENETIC
VARIATION ON DEVELOPMENT AND DISEASE
22.3.2.1. Most Xenopus Laboratory Populations
Are Genetically Diverse
Genetically diverse, outbred Xenopus populations have
been the traditional source of a rich history of biomedical
research. Their genetic diversity derives from three primary factors: outbred commercial stocks, random matings
to obtain experimental offspring, and design strategies to
increase diversity within an experimental group.
Most laboratories using Xenopus do not establish their
breeding colony by growing up the embryos produced
by the colony. Instead, they house colonies of up to several hundred sexually mature individuals purchased from
commercial frog breeders, such as Xenopus 1, Xenopus
Express, and Nasco. These commercial suppliers maintain
large populations of outbred adults that are sold to research
laboratories. Xenopus 1 maintains both a wild-caught and
a closed laboratory-bred colony, which are housed in separate facilities. For their laboratory-bred population, which
numbers approximately 20,000 including tadpoles, they
introduce about 1000 embryos from the wild-caught colony
every two to three years. These embryos are grown and bred
into the existing laboratory-bred colony to maintain genetic
diversity. Their wild-caught colony is a steady-state population of about 4500 adults replenished with f ve imports
from Chile per year; Xenopus were apparently introduced
into Chile in the 1950s (R. Weymouth, Xenopus 1, personal communication). The Nasco laboratory-bred colony
consisted of about 30,000 adults that were derived from
wild-caught founding adults imported from South Africa
between the early 1970s and 1996 (D. Brattlie, Nasco, personal communication). Although the Xenopus 1 and Nasco
populations are “closed,” their enormous sizes minimize
inbreeding and maximize genetic diversity. In both facilities, mating is random, producing laboratory-bred progeny
that are sold to research laboratories as well as added to the
breeding colony when they mature. Wild-caught animals
can be purchased from Xenopus 1 and Xenopus Express.
These animals are particularly favored by the research
community that uses oocytes for expression studies and
electrophysiology. Wild-caught animals are considered
more robust and disease resistant due to growing up in a
natural environment and are quarantined and tested for
pathogens before sale.
Typically, an individual laboratory maintains its breeding
colony of commercial and/or stock center acquired-adults to
produce offspring for experiments. Because the adults reproduce for up to 15 years, the colony within a single laboratory
often is composed of frogs that were purchased over many
years from more than one source. Accordingly, the adult
frogs in a given laboratory breeding colony are expected to
be unrelated and to display a high degree of genetic diversity, as shown by extensive EST and cDNA sequencing
(Blackshear et al., 2001; Fierro et al., 2007; Gilchrist et al.,
2004; Gilchrist and Pollet, 2012; Hellsten et al., 2007; Klein
et al., 2006). Even the comparison of RNAseq and genome
data between the inbred J and B strains revealed a degree of
sequence variation that resembled the situation determined
from comparing human genomes (Savova et al., 2017). Since
the typical commercial and laboratory breeding schemes
maintain a high level of genetic diversity in the offspring,
Xenopus has been proposed to be an ideal model to study
the functional relevance of specifc gene variants (Savova
et al., 2017).
The most common practices for generating offspring also
add genetic diversity to individual experiments. Typically,
embryos are produced by either natural mating of randomly
selected male and female pairs or by artif cial insemination
using the sperm of one randomly selected male to fertilize eggs from several randomly selected females. Because
embryos from different clutches might respond differently