Mapping a blood pressure quantitative trait locus to a 5.7-cM region
in Dahl salt-sensitive rats
Julie Dutil, Alan Y. Deng
Research Centre-CHUM, Hotel Dieu, 7-132 Pavillon Jeanne Mance, 3840 rue St. Urbain, Montre´al, Quebec, H2W 1T8, Canada
Received: 14 December 2000 / Accepted: 18 January 2001
Abstract. A region on rat Chromosome (Chr) 2 of the Dahl salt-
sensitive rat (S) was shown previously to contain a quantitative
trait locus (QTL) for blood pressure (BP). This was achieved first
by linkage, followed by the use of congenic strains. A congenic
strain, designated S.MNS-D2Mit6/Adh, contained a segment of
Chr 2 from the Milan Normotensive (MNS) rat in the S genetic
background. Since the region containing the QTL was roughly 80
cM in size, a further reduction was needed towards the positional
or candidate gene cloning. Currently, two congenic substrains
were made from the original strain S.MNS-D2Mit6/Adh. One of
these two substrains showed a BP-lowering effect, whereas the
other substrain did not. Deducing the segment not shared in the
two substrains, the BP QTL has to be present in a chromosome
region of roughly 5.7 cM between the marker D2Rat303 and the
locus for the neutroendopeptidase gene (Nep). Nep is not included
within the segment. This region does not seem to contain any
candidate genes well known for the BP control. Thus, the final
identification of the QTL will most likely lead to the discovery of
a brand new gene for the BP regulation.
The mapping of quantitative trait loci (QTL) for blood pressure
(BP) using animal models has been greatly facilitated by the ge-
netic approach, such as linkage analysis and the use of congenic
strains (Rapp and Deng 1995; Deng 1998; Dominiczak et al.
2000). In our previous work, regions on rat Chr 2 were shown to
contain QTL, first by linkage analysis (Deng and Rapp 1992; Deng
et al. 1994), followed by the use of two congenic strains (Deng et
al. 1997a). In one congenic strain, the chromosome region of in-
terest from the Dahl salt-sensitive strain (S) was replaced by that
of the Milan normotensive (MNS) rat (Deng et al. 1997a). The
length of the region involved in making the congenic strain was
about 80 cM, because the linkage evidence suggested (Deng et al.
1994) that one QTL was near the locus for the Na
subunit ␣ gene (Atp1a1) and another near the locus for the angio-
tensin receptor AT1B gene (Agtr1b)inanF
(S × MNS) popula-
tion. These two QTL could be separated by more than 40 cMs.
To further narrow down the region containing the QTL, we
made congenic substrains from the original strain, S.MNS-
D2Mit6/alcohol dehydrogenase (Adh) (Deng et al. 1997a), and
studied blood pressure of these substrains.
Materials and Methods
The SS/Jr rat used for making congenic substrains was provided
by J. Rapp and will be designated as S in the present report. The genomic
DNA for every S rat used for maintaining the strain and BP studies at each
generation has been extracted and verified by genotyping. In order to insure
that the S strain remains as genuine as the SS/Jr rat directly obtained from
J. Rapp in our facility, a rigorous and strict quality control procedure has
been instituted. It consists of two parts: (a) genetic testing; (b) physical
distinction. In an approximate genomic scan, 88 markers roughly evenly
spaced (on average 10–15 cM) throughout the rat genome were tested.
Each DNA sample for every S rat tested was compared with the true S
DNA standard that we used in our original work (Deng et al. 1997a). So
far, no marker differences have been detected (data not shown). Moreover,
all our S rats have, in addition to a skin tag, an ear mark punch hole
designed to facilitate their identification and to reduce potential handling
The original congenic strain used to initiate the study is designated as
S.MNSD2Mit6/Adh and is the same as published previously (Deng et al.
1997a). In brief, it was made by eight consecutive backcrosses and con-
sequently putting the MNS chromosome region between D2Mit6 and Adh
markers on the S genetic background. This strain, therefore, is homozygous
MNS (i.e., MM) for the region in question and homozygous SS for the rest
of the genome. This conclusion has been supported by the genotyping of 57
markers scattered throughout the rat genome other than on Chr 2 (data not
shown). Figure 1 contains the Chr 2 map, additional markers tested for the
Chr 2 regions, and the chromosome fragment in question in the congenic
strain. This original congenic strain is abbreviated as S.M. The authenticity
of the strain has been established by genotyping the markers between
D2Mit6 and Adh on Chr 2 (Fig. 1).
Protocols for handling as well as maintaining animals were approved
by our institutional animal committee. All the procedure for the experiment
was in accordance with the guidelines of local, provincial, and federal
Breeding scheme for generating substrains.
Rats of the original con-
genic strain, S.M (Fig. 1) were first bred with S to produce F
in turn were intercrossed to produce F
rat with crossovers in the
region between D2Mit6 and Adh markers was retained, and then back-
crossed (BC) to an S rat to duplicate the region of interest. A female and
a male BC rat were crossed to finally generate rats homozygous MM for
the region of interest, but homozygous SS for the rest of Chr 2 and rest of
the genome. The progeny of each of these crosses constitute a congenic
substrain dividing the initial segment involved in S.MNS-D2Mit6/Adh.
Substrains S.MNS-D2Mit6/D2Rat166 and S.MNS-D2Mit6/D2Rat303 are
abbreviated as S.M1 and S.M2 respectively (Fig. 1). The chromosome
region homozygous MM in the substrain and the original S.M strain are
shown as solid bars in Fig. 1. All the markers in the region were genotyped
for each congenic strain in question.
Preparation of rats for BP measurements.
The mating pairs for the S
strain and congenic strains to be studied were bred simultaneously and in
the same facility. Male rats were chosen from two separate litters of the
same strain when possible, in order to minimize potential litter effects. The
chosen rats were weaned at 21 days of age, maintained on a low-salt diet
(0.2% NaCl, Harlan Teklad 7034), and then fed a high-salt diet (2% NaCl,
Harlan Teklad 94217) starting from 35 days of age until the end of the
Telemetry probes were implanted when rats were 56 days old with their
body weights between 250–320 grams. Before the surgery, the rats were
anesthetized by the inhalation of isoflurane at a dose of 4% for 3 min.
During surgery, the state of anesthesia was maintained by isoflurane at a
Correspondence to: A. Deng; E-mail: email@example.com
Mammalian Genome 12, 362–365 (2001).
© Springer-Verlag New York Inc. 2001