The absence of mitochondrial DNA diversity among common laboratory inbred mouse strains
1 Department of Thoracic Surgery, Xinqiao Hospital, The Third Military
Medical University, Chongqing 400037, China
2 Center of Laboratory Animal, The Third Military Medical University,
Chongqing 400038, China
* Author for correspondence (e-mail: minjiaxin8{at}yahoo.com.cn)
Accepted 5 October 2005
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Summary |
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Key words: inbred strain, mouse, mtDNA, PCR-RFLP, PCR-SSCP, polymorphism
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Introduction |
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Most of the classical inbred mouse strains commonly used in biomedical
research descend from the colonies of a single mouse breeder, Abbie Lathrop of
Granby, MA, USA, in the early 20th century
(Beck et al., 2000). These
colonies were largely derived from European `fancy' mice (derivatives of the
domesticus subspecies of Mus musculus) and East Asian
`fancy' mice (derivatives of castaneus, molossinus and
musculus subspecies). It has long been recognized that because of
this unique man-made bottleneck, the genomes of these inbred strains originate
from a mixed but very limited pool of founders from the various subspecies
(Ferris et al., 1982
;
Bonhomme, 1987; Tucker et al.,
1992
; Ideraabdullah et al.,
2004
).
T739, 615 and TA2 are the main inbred mouse strains in China and are
recognized on a worldwide scale. These strains differ substantially from
classical strains such as BALB/c, C3H, C57BL/6J and DBA2, which were
established more than 60 years ago and are also designated as `old inbred'
strains in terms of anatomy, behavior and protein structure
(Ferris et al., 1983;
Zhang et al., 1998
). The TA2
strain originated from `Kunming' mice outbred at Beijing Bioproduct Institute
in 1962, and has been inbred since then. The T739 strain was derived from the
mating of a female `Kunming' mouse with a male 615 mouse, followed by sib
mating [data from the Mouse Genome Database (MGD)] at the Mouse Genome
Informatics Web Site of The Jackson Laboratory, Bar Harbor, MA, USA;
http://www.informatics.jax.org.
However, little is known about the phylogenetic relationships and recorded
origin of these strains.
Mitochondrial DNA (mtDNA) is a short circular molecule that, with the
exception of viruses, represents the most economically packed form of DNA in
the whole biosphere (Anderson et al.,
1981). The rapid evolution and maternal inheritance of mtDNA make
it a valuable marker for progeny of a given mother (Brown et al., 1979;
Hecht et al., 1984
;
Finnila et al., 2000
). In the
present study, we investigated mtDNAs from four classical (BALB/c, C3H,
C57BL/6J and DBA/2) and three Chinese (TA2, 615 and T739) mouse strains to
determine the phylogenetic relationships among these strains. Although it has
been known for many years that the four classical strains share a common
maternal origin, the question of whether the Chinese strains are of the same
origin has still to be answered, and is of interest for the study of
laboratory animals.
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Materials and methods |
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PCR amplification
The primers were synthesized by Shanghai Institute of Cell Biology
(Table 1). mtDNAs of mouse
liver cells were prepared by nuclei/cytoplasm partitioning
(Dai et al., 2000).
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PCR reactions were done in a volume of 100 µl and included 0.5 µg mtDNA as template. Amplification in the PCR consisted of an initial denaturation step at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 1 min, and extension at 72°C for 1 min. Following the final cycle, the mixture was incubated at 72°C for 10 min.
PCR-RFLP analysis of the D-loop, tRNAMet+Glu+Il and ND3 gene fragments
Two units of restriction endonucleases and 1.2 µl of 10x buffer
were added to 5 µl of PCR products, and then appropriate volumes of
sterilized water were added until the total volume reached 12 µl; the
mixture was then incubated at 37°C overnight. After digestion, each sample
was subjected to 1% gel electrophoresis with TBE buffer
(Dai et al., 1999). Following
electrophoresis at 3 V cm1 for 12 h, the resultant
gel was observed under ultraviolet and gel images were obtained.
PCR-SSCP analysis of D-loop 5' and 3' end fragments
PCR products were denatured with TE buffer
(Dai et al., 1999; diluted
1:10, pH 8.0). 5 µl of each diluted solution was mixed with 5 µl of
deionized formamide solution containing 20 mmol l1 EDTA,
followed by incubation at 95°C for 5 min. After chilling on ice for 10
min, samples were subjected to 9% polyacrylamide gel electrophoresis
(acrylamide:bisacrylamide = 49:1 v/v, containing 5% glycerol in 0.5 x
TBE). The electrophoresis was performed at a constant voltage of 25 V for
35 h under a controlled gel temperature of 20°C, and visualized by
silver staining.
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Results |
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Single-stranded conformation polymorphisms in D-loop 5' and 3' end fragments
To further analyze the genetic variations in these strains, D-loop 5'
and 3' end fragments, both of high variability, were subjected to
PCR-SSCP analysis. No differences in the SSCP electrophoresis bands were
observed (Fig. 2).
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Discussion |
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Owing to reactive oxygen species (ROS)-enhanced aggression, high
sensitivity to damage, and deficiency in repair of lesions, the rate of
sequence evolution in mtDNA is 1020 times higher than that in the
nuclear genome and, consequently, any two mtDNAs may differ by 1066
nucleotides (nt). The variability of different functional regions in mtDNA is
as follows (from least to greatest): ribosomal RNA, transfer RNA, known
proteins and displacement loop (Zeviani et
al., 1998; Chinnery et al.,
1999
). These mutations have accumulated sequentially along
radiating maternal lineages and now characterize animal populations in
different geographical regions of the world.
Restriction fragment length polymorphism (RFLP) or restriction maps
obtained by digesting mtDNA with restriction endonucleases can both be endowed
with species stability and reflect the relationship among species. mtDNA RFLP
is an efficient genetic marker with which to identify different animal species
and to infer the phylogenetic relationships among species; thus, analysis of
mtDNA RFLP has been extensively applied in the study of animal origination and
genetic differentiation (Brdicka et al., 1994; Zhang et al., 1992;
Oleinik et al., 2003). Ferris
et al. (1983
) analyzed mtDNA
RFLPs in more than 140 wild mouse types collected from all around the world
and in some `newly' inbred mouse strains (including MOR, PAC and SF/Cam /J).
They found many polymorphisms in fragment patterns and high levels of
variations in about 200 out of the 300 restriction sites studied.
Prior to the use of PCR techniques, analysis of mtDNA polymorphism involved
the isolation of mtDNA from cellular homogenates by separating nuclei and
cytoplasmic organelles, subsequent mtDNA purification by preparative
centrifugation in sucrose gradients, and then the search of RFLPs. With the
advent of PCR techniques, the analysis has become easier: the isolation of
mtDNA from total DNA is carried out by mitochondrion-specific primers rather
than biophysical methods, and the amount of tissue used to extract DNA
decreases from g to a few µg or even ng. In this study, the genetic
variations in mtDNA among the four classical (BALB/c, C3H, C57BL/6J and DBA2)
and the three Chinese (TA2, T739 and 615) strains were analyzed by PCR-RFLP
technique. Our findings showed no differences in the 46 restriction sites of
mtDNA D-loop, tRNAMet+Glu+Ile and ND3 gene fragments from these
strains, which is in agreement with the RFLP results for old inbred mice
(Ferris et al., 1983 and
Wang et al., 1992
).
A set of enzymes used in the RFLP analysis allows about 20% of the mtDNA
sequence to be examined (Wallace,
1994); therefore, a number of polymorphisms may remain undetected.
In recent years, different methods based on gel electrophoresis of PCR
products have been developed for detecting single-base mismatches in DNA,
including SSCP analysis (Thomas et al.,
1994
), denaturing gradient gel electrophoresis
(Gross et al., 1994
), and
mutation detection enhancement gel matrix
(Alonso et al., 1996
;
Finnila et al., 2000
). In
addition, sequencing the variable regions of mtDNA can ensure that a mutation
is not missed. In this way, Prager et al.
(1996
) were able to detect
differences between house mice that were indiscernible by other methods. A
newly developed program, Mutation Quantifiercan, accurately detect mutations
with frequencies as low as 3% (Song et
al., 2005
). In this study, to know more about the genetic
variations in these inbred strains, we analysed mtDNA D-loop 5' and
3' end fragments (both of high variability) by the PCR-SSCP technique.
PCR-SSCP analysis, first described in 1989, has been established as a
reliable, simple and fast method with high sensitivity and specificity for
detecting point mutations and sequence polymorphisms in short PCR-fragments up
to 350 bp. In PCR-SSCP, single-base-pair differences lead to varying mobility
patterns on nondenaturing polyacrylamide gels, as a result of altered
secondary and tertiary structures of DNA. Compared with direct sequencing,
PCR-SSCP analysis can reach a sensitivity (i.e. the ability to detect defined
point mutations) of 98% (Jaksch et al.,
1995
). Nevertheless, we found no variations in the SSCP
electrophoretic band pattern in these strains.
In view of enormous polymorphisms in mtDNA among mice and dramatic
differences in nuclear genomes of these seven inbred strains, our findings
were surprising (Nadeau et al.,
1981; Zhang et al.,
1998
; Wiltshire et al.,
2003
; Yalcin et al.,
2004
). Compared with nuclear DNA, mtDNA is characterised by faster
evolution and more polymorphisms. In mammals, the variation rate of mtDNA
sequence is about 1% (Upholt et al., 1977; Brown et al., 1981). However, the
absence of mtDNA variations contrasts sharply with the presence of large
nuclear differences in these strains we studied. This finding may be explained
by a close kinship and narrow genetic background
(Dai et al., 1999
). It was
reported that all of the `old inbred' strains (BALB/c, C3H, C57BL/6J, DBA2,
C58/J, SWR/J, AU/SsJ, PL/J and AKR/Cum), unlike wide or wild-derived inbred
mouse strains, had an identical mtDNA restriction endonuclease fragment
pattern, suggesting a single maternal lineage
(Ferris et al., 1982
). The
nine `old inbred' strains are thought to stem from a minimum of five different
female mice, which founded the primary strains. If these five females had been
picked at random from wild strains, the probability that all of them shared a
common type of mtDNA would have been extremely low
(
107; Ferris et
al., 1982
). However, most or all of them came from the pet mouse
trade, where the `old inbred' type of mtDNA could already have been present at
a high frequency. Non-random sampling, selective advantage and interstrain
contamination are three possible explanations that all the `old inbred'
strains contain a common type of mtDNA and thus appear to be of a single
maternal lineage.
In the present study, we corroborate the conclusion by Ferris et al.
(1982) using RFLP analysis.
Moreover, we suggest that the TA2, T739 and 615 strains, although established
in China, share a common maternal lineage with `old inbred' strains. The
polymorphisms in nuclear DNA among these strains are caused primarily by
mating with different males. This conclusion is also consistent with the
recorded origin of the inbred strains. Many of the strains are related, have
come from a common outbred colony, or have a common ancestry or other form.
For example, the TA2 strain originated from `Kunming' outbred `Swiss' strain
obtained from Haffkin Medical Science Institute of India in 1946
(Wang et al., 1992
); the 615
strain was derived from a `Kun Ming' mouse mated with a C57BL/6 mouse (Li et
al., 1989) and the T739 strain was derived from a cross between an albino
`Kunming' female mouse with a male mouse of 615 strain, followed by sib
mating, in 1973. The `Swiss' mice from mainly commercial sources, inbred in
different laboratories, are the origin of many inbred strains. For example,
SWR mouse, an `old inbred' strain, originated from Swiss mice inbred by Lynch
since 1926 (data from the Mouse Genome Database at the Mouse Genome
Informatics Web Site, The Jackson Laboratory, Bar Harbor, ME, USA;
http://www.informatics.jax.org).
Therefore, the TA2, T739 and 615 strains, as well as the `old' strains such as
BALB/c, DBA and C57BL, originated from relatively closed and
population-limited pet mouse colonies, and the chances are that these inbred
strains are of a common maternal lineage.
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