Brazilian Journal of Medical and Biological Research (1999) 32: 267-273 267

Characterization of rat strains by mtDNA RFLP

ISSN 0100-879X

Characterization of six rat strains

(Rattus norvegicus) by mitochondrial
DNA restriction fragment length
polymorphism

A.W. Hilsdorf 1,2 1Laboratório de Genética e Cardiologia Molecular,

and J.E. Krieger1 Instituto do Coração and Departamento de Clínica Médica,
Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil
2Departamento de Ciências Agrárias, Universidade de Taubaté, Taubaté, SP, Brasil

Abstract

Correspondence Restriction fragment length polymorphism (RFLP) was used to exam- Key words
J.E. Krieger ine the extent of mtDNA polymorphism among six strains of rats · Restriction fragment length
Laboratório de Genética e (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar polymorphism (RFLP)
Cardiologia Molecular
Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has · Rattus norvegicus
Instituto do Coração, HC, FM, USP mtDNA
Av. Dr. Enéas C. Aguiar, 44
revealed a low level of genetic divergence among strains. The sites of ·
· Laboratory rats
05403-000 São Paulo, SP cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic.
· Genetic monitoring
Brasil The use of these three enzymes allows the 6 strains to be classified into
E-mail: [email protected] 4 haplotypes and identifies specific markers for each one. The percent-
age of sequence divergence among all pairs of haplotypes ranged from
Research supported by FAPESP 0.035 to 0.33%, which is the result of a severe population constriction
(No. 4668-6), CNPq (No. 520696/95-
undergone by the strains. These haplotypes are easily demonstrable
6) and FINEP (No. 66.93.0023.00).
A.W. Hilsdorf is the recipient of
and therefore RFLP analysis can be employed for genetic monitoring
a Doctoral fellowship from CNPq to of rats within animal facilities or among different laboratories.
develop graduate studies in the
Department of Physiology and
Biophysics, IB-UNICAMP. Introduction This process was repeated in other laborato-
ries and today several strains of hypertensive
Rats are useful models employed to study rats with slightly different characteristics exist
Received July 1, 1998 cardiovascular physiology and to understand (3).
Accepted November 19, 1998 complex pathological states such as hyper- Several studies on genetic variability and
tension and diabetes mellitus (1). Rattus phylogeny in rodents based on mitochon-
norvegicus is the rodent species that gave drial and nuclear DNA have been published
rise to all types of laboratory rat strains cur- (4-6). At present, anonymous markers
rently in use. In the late fifties and early throughout the rat genome are available for
sixties several strains of rats were developed DNA “fingerprinting” (7,8). Restriction frag-
for high blood pressure studies by selective ment length polymorphism (RFLP) analysis
intercross to fix a phenotype among indi- of the nuclear genome was used to measure
viduals of outbred colonies. For instance, the the level of genetic divergence between SHR
spontaneously hypertensive rat (SHR) was and the Wistar-Kyoto rats (9). Also, arbi-
obtained by intercrossing individuals of an trarily primed polymerase chain reaction (AP-
outbred colony of Wistar Kyoto (WKY) rats PCR) was performed to develop nuclear ge-
with the highest blood pressure values (2). netic markers and to perform genetic linkage

Braz J Med Biol Res 32(3) 1999

268 A.W. Hilsdorf and J.E. Krieger

analysis in rat strains (10). More recently, monly used in the laboratory were investi-
Jacob et al. (11) used a large panel of mark- gated: Wistar, Wistar Munich, Brown Nor-
ers to develop a genetic linkage map for rats. way, Wistar Kyoto, SHR, and SHR-SP (spon-
The power of these methods is highlighted taneously hypertensive rat - stroke prone).
by their use in studies to analyze complex Male and female rats of 200 g were obtained
traits such as high blood pressure and diabe- from the animal facility at the Federal Uni-
tes mellitus (12-14). However, these tech- versity of São Paulo. Ten animals from each
niques are complex and are not available in strain were sampled from different matings.
most animal facilities where genetic moni- All procedures followed the “Guide for the
toring is undertaken. care and use of laboratory animals” (DHEW
Mitochondrial DNA diversity is known Publication No. (NIH) 85-23, Revised 1985,
to exist within the same species in different Office of Science and Health Reports, DRR/
taxa such as primates (15), fishes (16), and NIH, Bethesda, MD 20892) and the guide-
birds (17). Intraspecific mtDNA polymor- lines of the animal welfare act.
phism in Rattus norvegicus strains was ob-
served using the endonuclease EcoRI. The Mitochondrial DNA isolation
fragment pattern produced by EcoRI permit-
ted the classification of different rat strains Mitochondrial DNA was extracted from
into two categories, type A and type B (18,19). 3-4 g of liver and heart from each rat after an
Brown and Simpson (20) and Hayashi et al. overnight fast in order to decrease the glyco-
(21) found an extensive intra- and interspe- gen content in the liver. The tissues were
cific mtDNA polymorphism using not only homogenized in TEK buffer (50 mM Tris, 10
EcoRI but also HhaI, HindII, Hinf III, Hinf I mM EDTA, 0.2 M KCl, 0.25 M sucrose, pH
and HaeIII restriction endonucleases. 7.5) and the mtDNA was isolated according
Maternal mode of inheritance and lack of to the method of Chapman and Powers (24).
recombination of mtDNA are features that
can be used to obtain information about the Restriction endonuclease digestion
female genealogy and to organize the indi-
viduals into matriarchal lineages. Due to the The isolated mtDNA was digested with
ease of genotyping, mtDNA polymorphism 26 restriction endonucleases: ApaI (GGGCC/
may be a useful genetic marker to monitor C), AvaII (G/GWCC), BamHI (G/GATCC),
inbred lines in a variety of laboratories BglII (A/GATCT), BssHII (G/CGCGC), ClaI
(22,23). (AT/CGAT), DraI (TTT/AAA), EcoRI (G/
In the present study, a broad mtDNA AATTC), EcoRV (GAT/ATC), HincII (GTY/
survey of 6 strains of Rattus norvegicus was RAC), HindIII (A/AGCTT), HhaI (GCG/C),
carried out by RFLP. In addition, we estab- HpaI (GTT/AAC), KpnI (GGTAC/C), MluI
lished the pattern of genetic divergence a- (A/CGCGT), NcoI (C/CATGG), NdeI (CA/
mong these commonly used rat strains within TATG), NheI (G/CTAGC), PstI (CTGCA/
haplotypes that can be easily employed for G), PvuII (CAG/CTG), SacI (GAGCT/C),
genetic monitoring of animal facilities utiliz- SalI (G/TCGAC), SmaI (CCC/GGG), XbaI
ing three restriction endonucleases. (T/CTAGA), XhoI (C/TCGAG), and XmnI
(GAANN/NNTTC). mtDNA was digested
Material and Methods and incubated at 37oC for 3-4 h for complete
digestion as specified by the manufacturer
Experimental animals (Gibco, BRL, Gaithersburg, MD). The reac-
tion products were separated by electropho-
Six strains of Rattus norvegicus com- resis in 0.8% horizontal agarose gel and

Braz J Med Biol Res 32(3) 1999

Characterization of rat strains by mtDNA RFLP 269

visualized under UV light following ethi- XmnI, NcoI, and EcoRI, were successful in
dium bromide staining. The images were demonstrating differences in cleavage pat-
printed or stored for future analysis (Eagle terns which were considered to be polymor-
Eye System II, Stratagene). phic.
EcoRI proved to be the most informative
DNA size estimation enzyme with three different cleavage pat-
terns found in the 6 strains studied, as previ-
The mobility pattern of the mtDNA frag- ously shown. The Wistar strain mtDNA pre-
ments was compared to that of the standard sented 6 fragments, which correspond to
marker lambda HindIII and of the 100-bp type A (a), and Wistar Munich showed 4
DNA ladder (Gibco, BRL). Differences in fragments, described as type B (ß) (18,19).
fragment mobility were measured directly Wistar Kyoto, SHR, SHR-SP, and Brown
from the gel image and the DNA size was Norway rats showed a different pattern that
estimated using the DNAGEL software (25). is cited as type III by Brown and Simpson
(20) (Figure 1).
Data analysis Two new polymorphic sites were de-
tected during the present survey. First, NcoI
Percent sequence divergence among digestion revealed two restriction patterns.
mtDNA haplotypes was calculated accord- Wistar Kyoto, SHR, and SHR-SP mtDNA
ing to the following formula: d = [-loge S]/r. was cleaved into two similar size fragments,
For a class of endonucleases, r is the number whereas Wistar Munich, Wistar, and Brown
of nucleotides for a recognition sequence. S Norway mtDNA samples showed a single
= 2mxy/(mx + my) where mx is the number of site restriction fragment (Figure 2). The sec-
restriction sites for xth haplotypes, my the ond restriction enzyme, XmnI, also produced
number of restriction sites observed for the two restriction patterns. Wistar Kyoto, SHR,
yth haplotype, and mxy the common number SHR-SP, Wistar, and Brown Norway mtDNA
of restrictions between the xth and yth hap- samples showed three fragments while Wistar
lotype (26,27). A parsimony network was Munich mtDNA produced only two (Figure
constructed connecting the different haplo- 3).
types. Gains and losses at the network site The differences in cleavage patterns
are depicted as a single mutational step by a shown by HhaI (2 types) and HindII (2 types)
slash in the phenogram.
A 1 2 3 4 5 6 Figure 1 - EcoRI mtDNA cleav-
age pattern types of Rattus nor-
Results vegicus strains. Lane A, l HindIII
fragment; lane 1, cleavage pat-
tern I, Wistar; lane 2, cleavage
In order to develop a simple method to pattern II, Wistar Munich; lanes
monitor and test the use of mitochondrial 23.1
3-6, cleavage pattern III, Wistar
DNA as a genetic marker for laboratory rats, 9.4 Kyoto, SHR, SHR-SP, and Brown
Norway, respectively.
we performed a systematic survey using 26 6.6
4.4
restriction enzymes. Thirteen of them have
not been tested for this objective in pub- 2.3
2.0
lished studies. Three of the 26 endonucleases,
ClaI, NheI and SacI, were able to hydrolyze
the mtDNA from the 6 Rattus norvegicus
strains in only one site. Twenty endonu- 564
cleases cut the DNA twice or more, but did
not reveal polymorphism. Three enzymes,

Braz J Med Biol Res 32(3) 1999

270 A.W. Hilsdorf and J.E. Krieger

Figure 2 - NcoI mtDNA cleavage A 1 2 3 4 5 6 among several strains of R. norvegicus (28)

pattern types of R. norvegicus
strains. Lane A, l HindIII frag-
were not observed in the 6 strains presently
ment; lanes 1-3, cleavage pat- studied. The informative enzymes HinfII and
23.1
tern I, Wistar Kyoto, SHR, SHR- HaeIII utilized by Brown and Simpson (20)
SP, respectively; lanes 4-6,
9.4 were not used in our study since the large
cleavage pattern II, Wistar,
Wistar Munich, and Brown Nor- numbers of small size fragments produced
6.6
way, respectively. are difficult to visualize in ethidium bro-
4.4
mide-stained gels.
The analyses of the 6 strains with the
three polymorphic enzymes (EcoRI, XmnI
2.3
2.0
and NcoI) produced four haplotypes (Table
1). Wistar was designated as haplotype “A”;
Wistar Munich as “B”, Wistar Kyoto, SHR,
and SHR-SP as “C”, and Brown Norway as
“D”.
The estimated length of the mtDNA from
the 6 Rattus norvegicus strains averaged
16,500 ± 500 bp, which is in close agreement
with data previously published by Gadaleta
Figure 3 - XmnI mtDNA cleavage A 1 2 3 4 5 6
et al. (29) and Clark-Walker (30). Table 2
pattern types of R. norvegicus
strains. Lane A, l HindIII frag- shows the estimated mtDNA fragment sizes
ment; lanes 1-5, cleavage pat-
23.1 produced by each polymorphic enzyme.
tern I, Wistar Kyoto, SHR, SHR-
SP, Wistar, and Brown Norway,
A parsimony network was assembled
respectively; lane 6, cleavage
9.4 connecting the various clonal lineages ac-
pattern II, Wistar Munich. 6.6 cording to the pattern produced by the endo-
4.4 nuclease digestions (Figure 4). In the
phenogram one can estimate the relative ge-
netic distance by counting the number of site
2.3 differences among the haplotypes. There-
2.2
fore, intraspecific nucleotide diversity be-
tween all pairs of haplotypes can be calcu-
lated according to their rate of nucleotide
substitutions. Percent sequence divergence
(or index of nucleotide diversity) ranged
from 0.035 to 0.33% (Table 3).
The homogeneity of the strains studied
was tested by analyzing the pattern of mtDNA
fragments produced by EcoRI, NcoI and XmnI
Table 1 - Mitochondrial DNA clonal lineages of 6 strains of Rattus norvegicus based on
restriction sites produced by EcoRI, NcoI, and XmnI.
in 30 Wistar Munich rats from different
matings. No differences in restriction pat-
The Roman numerals indicate the cleavage pattern types for each enzyme. terns were found, indicating that the animal
Clone EcoRI NcoI XmnI Strains
colony tested is uniform.

A I II I Wistar Discussion
B II II II Wistar Munich
C III I I Wistar Kyoto, SHR, SHR-SP
Laboratory rats undergo a severe process
D III II I Brown Norway
of selection and inbreeding in order to pro-

Braz J Med Biol Res 32(3) 1999

Characterization of rat strains by mtDNA RFLP 271

duce isogenic lineages and the genetic stan- Table 2 - Fragment sizes (kb) produced by EcoRI, NcoI and XmnI restriction enzymes in
dardization required for experimental stud- mtDNA from Rattus norvegicus.
ies (31). Rats have been one of the most
frequently used animals to study cardiovas- The Roman numerals indicate the cleavage pattern types associated with each poly-
morphic enzyme.
cular physiology. Nonetheless, genetic vari-
ability has been identified in several strains EcoRI NcoI XmnI
of rats from different commercial sources as
I II III I II I II
well as from the same breeding facilities, a
fact that may influence interpretation of the 6.49 10.4 6.49 16.43 8.83 7.11 12.27
results (32). 4.11 3.05 4.11 7.60 5.06 4.04
3.05 2.54 3.05 4.04
Genetic variation in mtDNA is expected 1.87 0.46 2.54
to be small or even null since the process of 0.64 0.46
achieving inbred lines begins with a few 0.46
animals which possess the genetic character-
16.6 16.4 16.6 16.4 16.4 16.2 16.3
istics to be selected and fixed.
The various R. norvegicus types can be
regarded as different lineages due to the Figure 4 - Parsimony network
intensive inbreeding methods and also to the showing restriction site gains or

absence of gene flow among strains to pro- XmnI

B losses (slash in the diagram) in
Rattus norvegicus mitochondrial
duce rats for different experimental purposes. EcoRI DNA haplotypes. The DNA poly-
Consequently, the different strains may be EcoRI morphism is indicated on the
A D phenogram by its restriction en-
grouped into clonal lineages and their ge- donuclease.
netic variability assessed.
NcoI
As expected, our findings showed no C
differences among Wistar Kyoto, SHR and
SHR-SP strains upon analysis with the 26
enzymes tested, which is consistent with the
results found by Johnson et al. (9). This lack Table 3 - Matrix of percent sequence divergence
of divergence was expected since SHR and among the four haplotypes obtained by mtDNA
digestion with EcoRI, NcoI, and XmnI from 6
SHR-SP were developed from Wistar Kyoto strains of Rattus norvegicus.
rats by selection from a common maternal
lineage. The three polymorphic enzymes A B C D

identified in this work can easily be used to A - 0.33 0.14 0.1

classify Wistar Kyoto, SHR and SHR-SP B 0.33 - 0.25 0.22
into the same haplotype (C). C 0.14 0.25 - 0.035
Mitochondrial DNA from the Brown Nor- D 0.1 0.22 0.035 -

way strain exhibited the same pattern as found

in SHR, SHR-SP and Wistar Kyoto upon di- The three informative enzymes, EcoRI
gestion with EcoRI and XmnI. In contrast, and those identified in the present study,
NcoI produced a different cleavage pattern. XmnI and NcoI, can be used to separate the 6
Thus, Brown Norway rats were assigned to a rat strains into specific haplotypes with no
different haplotype (D). Wistar and Wistar need for sophisticated laboratory equipment.
Munich rats were classified as clones A and B, Various statistical approaches have been
which confirms the previously described types used to estimate mtDNA sequence diver-
A and B (18). These 2 strains were classified gence (33-35) and a range of results has been
differently upon digestion with the endonu- obtained. In the present study the nucleotide
cleases EcoRI and XmnI. diversity for R. norvegicus ranged from 0.035

Braz J Med Biol Res 32(3) 1999

272 A.W. Hilsdorf and J.E. Krieger

to 0.33%, which is lower than the values Wistar Munich rats originating from distinct
found by Brown and Simpson (20), 0.4 to mating pairs indicated a common founder
1.8%, and Hayashi et al. (21), 1%. The lower stock in the breeding facility studied. The
values of genetic divergence among the four origin of R. norvegicus plays an essential
clonal lineages obtained in our study can be role in the genetic background of the various
explained by the larger number of enzymes rat strains used in experimental studies.
surveyed. It is important to note that the final Therefore, the maintenance of genetic uni-
result was dependent on the analysis of all formity of laboratory rats depends on a care-
the informative and non-informative en- ful monitoring of the strains.
zymes. Additionally, the low divergence may The results obtained here suggest that the
be due to the severe population constriction analysis of mtDNA with EcoRI, NcoI, and
undergone by the strains which were gener- XmnI may be a useful tool to monitor labora-
ated from a small number of females. The tory rats, preventing strain contamination
highest sequence divergence value of 0.33% and maintaining population uniformity in
was found between haplotypes A (Wistar) animal facilities. The use of specific mtDNA
and B (Wistar Munich). This distance is probes and of non-invasive techniques (36)
illustrated in the parsimony network show- further improved the use of mtDNA for the
ing two restriction site losses for EcoRI and maintenance of the genetic quality of labora-
one for XmnI. Our results suggest that the tory rats. It is important to note that sorting
Wistar rat is the ancestral strain from which rat strain according to mtDNA clonal lin-
all the other strains studied originated. eages does not preclude a certain level of
It is interesting to note the low level of nuclear DNA polymorphism, which may be
divergence between haplotype D (Brown secondary to an outbred male colony, or to
Norway) and the others. This is consistent incomplete generation of inbreeding.
with a common and recent female origin of
the Brown Norway strain compared to the Acknowledgments
others even though the different coat colora-
tion of the Brown Norway strain indicates The authors gratefully thank Juliana S.
the contrary. A wild male rat probably intro- Valle, Maria E. Infante Vargas and Maria de
duced the dark coloration phenotype during Lourdes Junqueira for technical assistance
crossbreeding. and Dr. Ana Maria Lima de Azeredo Espin
The absence of differences in mitochon- for helpful discussion and suggestions.
drial DNA pattern among offspring from

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Braz J Med Biol Res 32(3) 1999