315
Natural Genetic Variation and Disease
effects of polymorphisms. Accordingly, genetic experiments
are usually performed in inbred animal lines, defned as the
result of at least 20 sequential generations of sister-brother
mating, to increase homozygosity.
22.2.1. MOUSE INBRED LINES
Inbred strains are the foundation of mouse developmental
genetics because they reduce genetic variability within and
across experiments performed in different laboratories.
Although many mouse strains used in current research are
close to homozygosity at any one locus, each strain has
unique sets of polymorphisms and modifers that affect
their responsiveness to genetic manipulations and treatments. As examples: C57BL/6J is considered optimal for
genetic engineering approaches, strain 129 is commonly
used to produce targeted mutations due to the availability
of many embryonic stem cell lines, and FVB is favored for
transgenic microinjections due to very large pronuclei and
large litter sizes (Blair et al., 2011; Bryant, 2011; Taketo
et al., 1991). The advantage of each strain, however, is
accompanied by different disease susceptibilities: C57BL/6
tends to develop age-related hearing loss, type 2 diabetes,
and atherosclerosis; 129 is susceptible to testicular teratomas; and FVB carries a mutation that results in blindness
(Bryant, 2011; Rashid et al., 2019; Stevens and Hummel,
1957). Thus, although the fxed genetic composition of
inbred lines makes them extremely useful for specif c
experimental manipulations, their underlying genetic constraints render them less than optimal for determining how
health-related manipulations will affect genetically variable human populations.
An additional caution is that there are sublines of the
common inbred lines that can be distinctly different at the
genomic level, such as multiple 129 substrains (Simpson
et al., 1997; Threadgill et al., 1997 ) or the two C57BL/6
substrains (6J, 6N), which have been separated for about
220 generations and differ by numerous SNPs, indels, and
SVs (Simon et al., 2013). In addition to substrain variability, there can be signifcant differences between mice
originating from the same substrain but housed within
different laboratory colonies (Justice and Dhillon, 2016).
Thus, an inbred mouse strain may not be as “isogenic” as
presumed.
22.2.2. COMMON FISH LINES
Medaka (Oryzias latipes) and zebraf sh (Danio rerio) are
the most common fsh used in biomedical research. Several
inbred medaka lines have been generated by sibling mating for over 20 generations, and several of these inbred
lines have heterozygosity levels that are nearly 100-fold
lower than in wild-caught fsh (Spivakov et al., 2014). In
contrast, zebrafsh are prone to “inbreeding depression,”
in which during the inbreeding process they exhibit a
decline in successful matings and clutch size and an
increase in offspring sterility (Monson and Sadler, 2010;
Shinya and Sakai, 2011). The two available lines considered inbred (C32, SJD) were derived by early-pressure egg
parthenogenesis (Johnson et al., 1995; Streisinger et al.,
1981). Similar to mouse, different zebrafsh strains are
genetically divergent from each other, from wild-caught
fsh from different localities, and from isolates of the
same strain kept in different laboratories (Balik-Meisner
et al., 2018; Franek et al., 2020; Suurväli et al., 2020). The
inter-lab genetic diversity is thought to result from using a
breeding laboratory population that is too small (Suurväli
et al., 2020).
22.2.3. AVAILABLE XENOPUS LINES
Xenopus is the most common amphibian model used for
biomedical research, and there are a few inbred lines. The
Xenopus laevis J strain originated from a laboratory in
Switzerland and was sent to the United States and then to
Japan (hence “J” strain). In 1992, the 21st generation no
longer exhibited long-term skin rejection, indicating that
they were extensively homozygous. A descendant from the
30th generation was used for genome sequencing (Session
et al., 2016), and subsequent generations were used to create BAC libraries (32nd and 33rd), RNAseq data (33rd and
34th), and chromosomal f uorescent in situ hybridization
mapping (33rd) (Session et al., 2016). Like other inbred
animals, the more uniform genetic background of J strain
frogs enables improved accuracy for genome editing techniques (e.g. Ratzan et al., 2017), and the homozygosity of
the MHC locus provides an important resource for immunological research (Gantress et al., 2003). The Xenopus
laevis B strain originated at the Institute of Developmental
Biology in Moscow as a spontaneous albino mutation,
periodic albinism (a p) ( Hoperskaya, 1975 ). Individuals
were imported to Berkeley in the 1980s, and as a result
of edemas, they were occasionally outbred to pigmented
frogs and then crossed back to obtain the albino phenotype
without edema (Savova et al., 2017). Since 1994, B strain
frogs have been interbred for approximately ten generations (Savova et al., 2017).
Xenopus tropicalis was introduced to biomedical research
because it has a simpler genome (diploid vs. allotetraploid)
and a shorter generation time (5–8 months vs. 6–12 months)
than Xenopus laevis. Beginning in the early 1990s, several
laboratories obtained wild-caught Xenopus tropicalis from
Nigeria (N) and the Ivory Coast (IC) (Grainger, 2012). The
Golden strain was produced by selecting N frogs for rapid
growth rate and early sexual maturity, resulting in a shorter
generation time (Horb et al., 2019). Three stock centers
(European Xenopus Resource Centre [EXRC, England],
National BioResource Center [NBRC, Japan], National
Xenopus Resource [NXR, USA]) produced eight Xenopus
tropicalis lines (5 N and 3 IC), and by 2015, three N lines
had achieved a high degree of homozygosity (Igawa et al.,
2015). Currently, N lines are at the 10th (NH), 11th (Golden,
BH), and 18th (NA) generations, and the IC strains are at
least at the 5th generation.
Natural Genetic Variation and Disease
effects of polymorphisms. Accordingly, genetic experiments
are usually performed in inbred animal lines, defned as the
result of at least 20 sequential generations of sister-brother
mating, to increase homozygosity.
22.2.1. MOUSE INBRED LINES
Inbred strains are the foundation of mouse developmental
genetics because they reduce genetic variability within and
across experiments performed in different laboratories.
Although many mouse strains used in current research are
close to homozygosity at any one locus, each strain has
unique sets of polymorphisms and modifers that affect
their responsiveness to genetic manipulations and treatments. As examples: C57BL/6J is considered optimal for
genetic engineering approaches, strain 129 is commonly
used to produce targeted mutations due to the availability
of many embryonic stem cell lines, and FVB is favored for
transgenic microinjections due to very large pronuclei and
large litter sizes (Blair et al., 2011; Bryant, 2011; Taketo
et al., 1991). The advantage of each strain, however, is
accompanied by different disease susceptibilities: C57BL/6
tends to develop age-related hearing loss, type 2 diabetes,
and atherosclerosis; 129 is susceptible to testicular teratomas; and FVB carries a mutation that results in blindness
(Bryant, 2011; Rashid et al., 2019; Stevens and Hummel,
1957). Thus, although the fxed genetic composition of
inbred lines makes them extremely useful for specif c
experimental manipulations, their underlying genetic constraints render them less than optimal for determining how
health-related manipulations will affect genetically variable human populations.
An additional caution is that there are sublines of the
common inbred lines that can be distinctly different at the
genomic level, such as multiple 129 substrains (Simpson
et al., 1997; Threadgill et al., 1997 ) or the two C57BL/6
substrains (6J, 6N), which have been separated for about
220 generations and differ by numerous SNPs, indels, and
SVs (Simon et al., 2013). In addition to substrain variability, there can be signifcant differences between mice
originating from the same substrain but housed within
different laboratory colonies (Justice and Dhillon, 2016).
Thus, an inbred mouse strain may not be as “isogenic” as
presumed.
22.2.2. COMMON FISH LINES
Medaka (Oryzias latipes) and zebraf sh (Danio rerio) are
the most common fsh used in biomedical research. Several
inbred medaka lines have been generated by sibling mating for over 20 generations, and several of these inbred
lines have heterozygosity levels that are nearly 100-fold
lower than in wild-caught fsh (Spivakov et al., 2014). In
contrast, zebrafsh are prone to “inbreeding depression,”
in which during the inbreeding process they exhibit a
decline in successful matings and clutch size and an
increase in offspring sterility (Monson and Sadler, 2010;
Shinya and Sakai, 2011). The two available lines considered inbred (C32, SJD) were derived by early-pressure egg
parthenogenesis (Johnson et al., 1995; Streisinger et al.,
1981). Similar to mouse, different zebrafsh strains are
genetically divergent from each other, from wild-caught
fsh from different localities, and from isolates of the
same strain kept in different laboratories (Balik-Meisner
et al., 2018; Franek et al., 2020; Suurväli et al., 2020). The
inter-lab genetic diversity is thought to result from using a
breeding laboratory population that is too small (Suurväli
et al., 2020).
22.2.3. AVAILABLE XENOPUS LINES
Xenopus is the most common amphibian model used for
biomedical research, and there are a few inbred lines. The
Xenopus laevis J strain originated from a laboratory in
Switzerland and was sent to the United States and then to
Japan (hence “J” strain). In 1992, the 21st generation no
longer exhibited long-term skin rejection, indicating that
they were extensively homozygous. A descendant from the
30th generation was used for genome sequencing (Session
et al., 2016), and subsequent generations were used to create BAC libraries (32nd and 33rd), RNAseq data (33rd and
34th), and chromosomal f uorescent in situ hybridization
mapping (33rd) (Session et al., 2016). Like other inbred
animals, the more uniform genetic background of J strain
frogs enables improved accuracy for genome editing techniques (e.g. Ratzan et al., 2017), and the homozygosity of
the MHC locus provides an important resource for immunological research (Gantress et al., 2003). The Xenopus
laevis B strain originated at the Institute of Developmental
Biology in Moscow as a spontaneous albino mutation,
periodic albinism (a p) ( Hoperskaya, 1975 ). Individuals
were imported to Berkeley in the 1980s, and as a result
of edemas, they were occasionally outbred to pigmented
frogs and then crossed back to obtain the albino phenotype
without edema (Savova et al., 2017). Since 1994, B strain
frogs have been interbred for approximately ten generations (Savova et al., 2017).
Xenopus tropicalis was introduced to biomedical research
because it has a simpler genome (diploid vs. allotetraploid)
and a shorter generation time (5–8 months vs. 6–12 months)
than Xenopus laevis. Beginning in the early 1990s, several
laboratories obtained wild-caught Xenopus tropicalis from
Nigeria (N) and the Ivory Coast (IC) (Grainger, 2012). The
Golden strain was produced by selecting N frogs for rapid
growth rate and early sexual maturity, resulting in a shorter
generation time (Horb et al., 2019). Three stock centers
(European Xenopus Resource Centre [EXRC, England],
National BioResource Center [NBRC, Japan], National
Xenopus Resource [NXR, USA]) produced eight Xenopus
tropicalis lines (5 N and 3 IC), and by 2015, three N lines
had achieved a high degree of homozygosity (Igawa et al.,
2015). Currently, N lines are at the 10th (NH), 11th (Golden,
BH), and 18th (NA) generations, and the IC strains are at
least at the 5th generation.