255
Rapid Communication
Analysis of the Genetic Contamination of
Salt-Sensitive Dahl/Rapp Rats
James L. Lewis, Robert J. Russell, David G. Warnock
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract Genetic contamination of Dahl/Rapp salt-sensitive rats (SS/JrHsd) was recently discovered in animals received from Harlan Sprague Dawley, Inc, the main supplier of
this strain to researchers in the United States. We were
interested in determining how this problem occurred and the
extent of contamination in the breeding colony in hopes of
quickly correcting the problem and reestablishing the supply of
this important model of genetic hypertension. DNA was
extracted from whole blood obtained from each rat in the
Harlan foundation colony and their offspring, the pedigree
expansion colony. Several microsatellite-based genetic markers that were polymorphic between the normal and contami-
nant alleles were used to test these two colonies. All 10
breeder pairs in the foundation colony were tested at six
different loci and found to be homozygous for the normal
allele in each case. All 60 members of the pedigree expansion
colony were also tested, and several rats carrying contaminant
alleles were found, thus localizing the origin of the contamination to this colony. We offer several recommendations
regarding precautions researchers using inbred animals should
take in designing future experiments. (Hypertension. 1994;24:
255-259.)
Key Words • blood pressure • hypertension, genetic •
rats, inbred strains • genetics
W
Earlier this year several different investigators working
with S and R rats from Harlan began to notice the loss
of the hypertensive phenotype in S rats placed on a high
salt diet (WJ Welch and CS Wilcox, personal communications). In a recent article, St Lezin et al15 detailed
the discovery of this problem.
We were interested in determining how this problem
occurred and the extent of contamination in the breeding colony in hopes of quickly correcting the problem
and reestablishing the supply of this important model of
genetic hypertension. We devised a plan to systematically search for the source of the genetic contamination
in the S rat breeding colony. As diagrammed in Fig 1,
the S rat breeding colony is currently maintained in
three separate levels. The first, self-propagating colony,
termed the foundation colony (FC), remains in barrier
pathogen-free conditions. At no time are rats introduced into this colony. Their offspring become breeders
in a second colony, the pedigree expansion colony
(PEC). The offspring of this colony pass on to the
production colony (PC), which supplies offspring for
distribution. There were 10 breeder pairs in the FC, 20
breeding trios (two females and one male) in the PEC,
and more than 200 breeders in the PC. Obviously,
genetic contamination could occur at any of these levels.
The rapid determination of the distribution of the
contaminant alleles was needed to expedite regeneration of the colonies and reestablishment of the supply of
purebred S rats.
ith the advent of the widespread application
of genetic techniques to biomedical research, inbred animal models of human disease have become important tools for use in the discovery of genes involved in disease processes showing
simple mendelian inheritance as well as those, such as
hypertension, that show porygenic patterns of inheritance. The inbred Dahl/Rapp salt-sensitive (SS/JrHsd
or S) and salt-resistant (SR/JrHsd or R) rats represent
one of the best characterized and most widely studied
models of salt-sensitive hypertension.12 The power of
the genetic paradigm, as applied to the study of hypertension, was first applied in this model by Rapp et al, 34
who found a cosegregation between a polymorphism on
the rat renin gene and hypertension in F 2 progeny of a
cross between these two strains. This approach has been
used by several groups to search for genes related to
blood pressure control in this and other rat models of
genetic hypertension.513 Careful maintenance of inbred
hypertensive rat strains is critical to work of this type.
Harlan Sprague Dawley, Inc (Indianapolis, Ind) has
maintained S and R rats with a program of strict
inbreeding since receiving these strains from Dr John
Rapp in 1986. By 1991, the inbreeding of both these
strains for more than 50 generations resulted in a very
high level of genetic homogeneity within each strain
and, as a result, very reliable physiological responses.14
Received January 17, 1994; accepted in revised form July 18,
1994.
From the Department of Medicine, Division of Nephrology and
Nephrology Research and Training Center, University of Alabama at Birmingham (J.L.L., D.G.W.); the Department of Veterans Affairs Medical Center, Birmingham, Ala (D.G.W.); and
Laboratory Animal Medicine, Harlan Sprague Dawley, Inc, Indianapolis, Ind (RJ.R.).
Correspondence to James L. Lewis, MD, The University of
Alabama at Birmingham, 618 Zeigler Research Bldg, 703 S 19th
St, Birmingham, AL 35294-0007.
© 1994 American Heart Association, Inc.
Methods
Experimental Animals
All studies were done in accordance with The University of
Alabama at Birmingham Institutional Animal Care and Use
Committee guidelines. Six commercially available 6-week-old
male rats (offspring of PC breeders) of both S and R strains
were obtained from Harlan Sprague Dawley in October 1993.
Two S rats that had been obtained from Dr John Rapp at the
Medical College of Ohio in August 1993 were used as S rat
256
Hypertension
Vol 24, No 3
September 1994
H«rttn
D»riY»aono<
SandR Stock*
John Rapp
luaDawtsy
M
[SS/JrHad and SWJiWsdl
Foundation
CotoniM
I
(10 bre«ter pairs) J
TABLE 1 . Genetic Markers Used to Screen for
Contaminant S* Alleles in Commercially Available
Dahl/Rapp Salt-Sensitive Rats
Locus (Marker)
Rat
Chromosome
S* Allele
Size
2
>S
•do, (Ma)
Guanylyl cyclase A
PadkjiM
Exprtaton
(20 mrin, 40 tomato)
DaNSandR
Outbrsd Stocks
Production
Colon kw
(200 breading pairs)
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
FIG 1. Diagram shows rough timeline and breeding colony
arrangement at Harian Sprague Dawley for satt-sensrttve (S)
Dahl/Rapp rats. R indicates salt-resistant; SS/JrHsd, Dahl/Rapp
salt-sensitive strain; and SR/JrHsd, Dahl/Rapp salt-resistant
strain.
controls. In addition, archived DNA samples from S rats
obtained from Harian for use in our laboratory (J.L.L. and
D.G.W.) before 1992 were used. Each S rat in the FC and PEC
at Harian was used to supply blood for DNA analysis.
Collection of Contaminated Rat DNA
S and R rats were anesthetized with pentobarbital (5.0
mg/100 g body wt IP) and killed by severing the thoracic aorta.
Genomic DNA was harvested using a modification of the
method of Blin and Stafford16 as follows: 1 g liver was excised,
frozen in liquid nitrogen, and crushed into a fine powder. This
was suspended in a digestion buffer (0.1 mg/mL proteinase K,
0.5% sodium dodecyl sulfate, 10 mmol/L Tris-HCl [pH 8], 25
mmol/L EDTA [pH 8], 100 mmol/L NaCl) and incubated at
50°C overnight. The resulting digest was extracted three times
with a Tris-buffered phenol (pH 8.0), chloroform, and isoamyl
alcohol mixture (25:24:1). DNA was then collected with ethanol precipitation. Purity and yield were assessed by measuring the optical density at 260 and 280 nm with a UV/Vis
spectrophotometer (Perkin-Elmer).
Development of Informative Genetic Markers
Polymerase chain reaction (PCR) primers amplifying areas
containing dinucleotide repeats (microsatellites) were used to
amplify genomic DNA from the six S and R rats obtained from
Harian in parallel with those obtained from J. Rapp and
archived S rat DNA samples from our laboratory. Several
markers were used as described by Jacob et al6 and Serikawa
et al 17 (MapPairs, Research Genetics). PCR was done in a
50-^L reaction volume containing 10 mmol/L Tris-HCl (pH
8.3), 50 mmol/L KC1,1.5 mmol/L Mg, 0.001% (wt/vol) gelatin,
200 /imol/L dNTPs, and 2.5 U Amplitaq DNA polymerase
(Boehringer Mannheim Biochemicals). Twenty picomoles of
each primer and 100 ng of rat genomic DNA were used. After
an initial melting period at 94°C for 5 minutes, the DNA was
amplified through 35 cycles of 94°Cxl minute, 55°Cxl
minute, and 72°Cxl minute, 30 seconds with an automated
thermal cycler (Perkin Elmer Cetus). PCR products were
separated on a 2% Nusieve 1:3 agarose gel (FMC Bioproducts), stained with ethidium bromide, and visualized by UV
transillumination. In addition, a primer pair that amplified a
microsatellite within the guanylyl cyclase A (GCA) gene was
used as described by Deng and Rapp. 8 PCR amplification and
size fractionation of the products were done as described
above.
(MIT-R721)
6
<S
Angiotensin-converting enzyme
10
=
Asialogtycoprotein receptor-1
10
=
Pancreatic polypeptide
10
=
Sex hormone binding globulin
10
=
ATP citrate lyase
10
=
>s
=
<s
>s
>s
(MIT-R354)
12
Renin
13
(MIT-R1041)
14
Transthyretin
18
Intestinal calcium binding protein
X
Allele size relative to the normal salt-sensitive (S) allele is
shown. No polymorphism between S and S* is denoted by =.
Collection of Genomic DNA From FC and
PEC Rats
Roughly 500 fiL whole blood was collected from each FC and
PEC rat into a buffered sodium citrate solution after light
anesthesia with inhaled carbon dioxide and shipped by overnight
delivery to the University of Alabama at Birmingham on ice. On
arrival, genomic DNA was extracted from each sample with
microprep spin columns (QIAamp, QIAGEN, Inc) using reagents supplied with the kit. Isopropanol (210 /xL) was substituted for ethanol before the digest was loaded onto the column,
and DNA was eluted from the columns with 200 /iL of 0.1 mol/L
Tris, pH 9.0, preheated to 70°C to maximize yield. Purity and
yield were assessed with a UV spectrophotometer (Pharmacia
Biotech, Inc). Samples were stored at 5°C until use.
Genotyping FC and PEC DNA
The DNA collected from each member of the FC and PEC
colonies was amplified with PCR at several different loci that
were found to be polymorphic between normal (S) and contaminant (S*) alleles. The FC was tested with markers at the
GCA, transthyretin (TTR), and intestinal calcium binding
protein (CBPI) loci as well as three other markers located on
rat chromosomes 6, 12, and 14 (Research Genetics MapPairs
nos. MIT-R721, MIT-R354, and MIT-R1041, respectively).
DNA from each member of the PEC was also amplified at the
GCA and MIT-R354 loci.
Results
Description of S* Alleles
Screening of S and R rats recently obtained from
Harian and J. Rapp as well as archived S rat DNA was
performed with those markers listed in Table 1. A single
allele was detected in each R rat with each marker (data
not shown). Six loci on different rat chromosomes were
polymorphic between S and S* alleles. Fig 2 shows PCR
products amplified with three such polymorphic markers. Table 2 lists the genotypes obtained on S rats from
a variety of sources with the six informative markers.
There is independent assortment of S* alleles in the S
rats recently obtained from Harian. No S* alleles were
found in rats obtained from Harian before April 1992 or
in those from J. Rapp.
Lewis et al
Genetic Contamination of Dahl/Rapp Rats
Since generation of a new FC would require rederivation of pathogen-free breeders involving either hysterectomy, embryo transplantation, or cross fostering
schemes, a great deal of time and expense could be
avoided if the FC was found to be unaffected. For these
reasons, the search for the source of genetic contamination focused on the FC and PEC. Our results show no
evidence of contamination in the FC with six informative markers dispersed throughout the rat genome. We
conclude that the S rat FC at Harlan is unaffected by the
genetic contamination affecting S rats available in 1993.
Genetic contamination was found in several members
of the PEC. After screening with two genetic loci, we
found that 45% of the rats in the PEC carried at least
one S* allele. The precise events leading to the genetic
contamination in this colony are not clear; however,
several other strains of inbred rats were housed in the
same room as the PEC. Because of the close proximity
of the PEC and these other strains, one or more non-S
rats may have been accidentally introduced into the S
rat PEC breeding colony. Since only a single contaminant allele was found, one might speculate that this was
another inbred strain. If this was the case, those offspring carried forward to the PC would be similar to an
F! cross, and their offspring would in turn be typical of
an F2 population. This would account for the independent assortment of alleles observed in S rats obtained
from Harlan at the onset of these studies. Screening of
the PEC is not consistent with this theory, however,
because heterozygotes were present, and independent
assortment was also observed. No single introduction of
a non-S rat into the PEC can explain this finding,
although it is possible to imagine a series of contaminations occurring between the S rat PEC and other strains
that would explain it. If, on the other hand, contamination occurred in the FC, one would expect to find traces
of the genetic contamination there because this colony
self-propagates. Since no S* alleles were found in the
FC, we conclude that the S rat breeding colony at
Harlan became genetically contaminated through the
accidental introduction of another rat strain at the level
of the PEC.
Given that the contamination occurred in the PEC,
the next question that needs to be addressed is how the
genetic contamination escaped detection. The inbred S
rat colony at Harlan has been monitored annually using
six biochemical markers (Hbb, Es-1, Es-2, Pgd, G.c,
Loci
RatChrom.
U
600 bp-
FIG 2. Ethidium bromide-stained agarose gel shows polymerase chain reaction products obtained by amplification of rat
genomic DNA templates. Data from three genetic markers are
shown. B indicates blank; lane 1, salt-sensitive (S) rat (obtained
before 1992); lane 2, salt-resistant (R) rat (obtained before
1992); lanes 3 and 4, contaminated S rats; and lane 5, S rat
(obtained before 1992). M=100 bp ladder.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Screening FC and PEC
The six informative markers were then used to amplify
genomic DNA from each member of the FC. Fig 3A and
3B show PCR products for each FC rat after amplification
with GCA (lanes 1 through 20). Testing of the FC was
carried out with each of the remaining five PCR primer
pairs with similar results (see Table 2). Fig 3B also shows
amplified products from 16 PEC rats demonstrating the
presence of SS, SS*, and S*S* individuals. In total there
were 35 SS, 12 SS*, and 5 S*S* genotypes in the PEC
(eight samples showed poor amplification). As shown in
Table 2, amplification of DNA from each PEC member
was done with two of the informative markers: GCA and
MTT-R354. Amplification with MTT-R354 showed the
presence of only SS and SS* genotypes in members of the
PEC. There was no relation between the genotype at this
locus and that at GCA.
Discussion
We have confirmed that genetic contamination of the
S rat breeding colony at Harlan Sprague Dawley did
indeed occur. S rat offspring of the present PC not only
show heterozygosity at several genetic loci but independent assortment of these alleles as well. This implies
that contamination occurred at least two generations
above these rats in the breeding pedigree. We suspected
that the problem had occurred in either the FC or PEC.
TABLE 2. Genetic Screening of Dahl/Rapp Salt-Sensitive Rat Colonies From Various Sources With
Six Genetic Markers
Genotype, Locus (Marker)
S Rat Source
GCA
(R721)
(R354)
TTR
(R1041)
CBPIt
J. Rapp (8/93)
SS
SS
SS
SS
SS
SS, S
ss
ss, ss*, s*s*
ss
ss, ss*, s*s*
ss
ss, ss*
ss
SS
ss
ss, ss*
ss
SS
ss, s
s, s*
ss, s
Harlan (late '91 /early '92)
Harlan (10/93)
FC
PEC
257
SS, SS*
SS
SS, SS*
ss
ss, ss*
S indicates salt-sensitive; GCA, guanytyl cyclase A; TTR, transthyretin; CBPI, Intestinal calcium binding protein;
FC, foundation colony; and PEC, pedigree expansion colony. In the Genotype columns, S shows the presence of
normal S allele; S*, presence of contaminant allele. Therefore, SS* shows heterozygote at locus in question.
tSince CBPI Is an X chromosomal marker, a single allele is present in males.
258
Hypertension Vol 24, No 3 September 1994
PECRats
A
M 1 2 3 4 5 6 7 8 910
12 14 16 18 U
11 13 15 17
600 bp-
M19 20
600 bp
FK3 3. Ettiidium bromide-stained agarose gels show potymerase chain reaction products obtained by amplification of foundation
colony (FC) rat DNA with primers specific for the guanytyi cyclase A (GCA) locus. A and B, lanes 1 through 20 are FC breeders from
Harlan. B, Products from a representative sample of 16 pedigree expansion colony (PEC) rats. The first two PEC lanes show individuals
heterozygous at the GCA allele (S,S*). The third PEC lane shows an individual homozygous for the S* allele at this locus. M = 100 bp
ladder.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
and Pep-3) and starting in 1992 DNA fingerprinting
techniques as previously described.18 Genetic monitoring was performed on some breeders from the FC but
not on rats from the PEC, PC, or the individual animals
distributed from the PC to investigators. This monitoring program failed to discover the genetic contamination under consideration. It is unknown whether the
DNA fingerprinting assay used would have uncovered
the problem had contaminated rats been tested. Since
the contamination did not affect the FC, no amount of
screening of this colony would uncover the problem.
Genetic screening at multiple levels within the colony
should improve the chances of finding future genetic
contamination. More frequent than annual monitoring
would also improve future genetic screening schemes.
Careful consideration should also be given to the selection of microsatellite markers included in a screening
panel. Markers scattered throughout the genome that
have been shown to be polymorphic between the strain
of interest and all other strains at the vendor should be
used if such markers can be found. A screening program
incorporating these elements may very well have identified the problem with the S rats at an earlier time and
minimized the effect of the incident.
The magnitude of work and time lost as a result of
this mishap leaves a clear message to those interested in
working with inbred rat models. Both the investigator
and the breeding facility must take great care to assure
the genetic purity of these strains because both the
physiological and genetic advantages of working with an
inbred strain are lost if mismating occurs. Several
conclusions can be made from the present data. First,
an inbred line can become genetically contaminated
quite easily, and every effort should be made to maintain strict, physical separation of these strains at the
breeding facility. Second, although it is impossible to
predict which genetic loci will be affected in a given
future contamination mishap, screening markers should
be chosen prospectivery to be highly polymorphic among
possible contaminant alleles. Despite the use of such
safeguards, there is no guarantee that an inbred strain
will never become genetically contaminated in the future. For this reason, archiving DNA or tissue (liver can
be successfully stored at -80°C for several years for
later DNA extraction) from each rat should be considered as a critically important responsibility of the individual investigator. Finally, the initial observation iden-
tifying this problem was the loss of the hypertensive
phenotype in S rats. In this case, the loss of hypertension
was a very sensitive marker of the genetic purity of the
hypertensive S strain. Several groups have shown that the
Fi and F2 offspring of a cross between S rats and another
inbred, normotensive strain have blood pressure phenotypes that are intermediate between the two parental
strains.2-6-7-13'19-20 Therefore, incorporating periodic blood
pressure monitoring into experimental protocols involving
inbred hypertensive rats is another important safeguard to
be considered. To preserve this extra safety measure,
vendors should take extreme precautions to avoid mismating between two hypertensive strains. Table 3 lists several
recommendations based on these observations for investigators planning work with inbred hypertensive rat
strains.
Based on these studies, the SS/JrHsd rat PEC and PC
at Harlan are being regenerated with offspring from the
FC. Given the average generation time of Rattus norvegicus, the commercial availability of genetically pure S
rats from this vendor will be reestablished in mid-1994.
Establishing that the offspring from the regenerating S
rat breeding colony once again demonstrate the hypertensive response to high salt diet as well as not carry the
contaminant alleles described above will be critical to
the confirmation that the progeny of the newly generTABLE 3. Recommendations for Researchers Working
With Inbred Hypertensive Rat Strains
1. Ascertain that strains are fully inbred (at least >20
generations).
2. Discuss with supplier their policy regarding maintenance of
inbred strains.
a. Are animals physically isolated from other strains at the
vendor?
b. Do inbreeding procedures include the use of foundation,
pedigree expansion, and production colonies?
c. What is the nature of the genetic monitoring program
performed by the vendor?
d. Is there a program of periodic phenotyping of the strain
at the vendor?
3. Incorporate periodic blood pressure monitoring into
experimental design.
4. Collect/archive specimens (liver at -80°C) for later DNA
analysis.
Lewis et al Genetic Contamination of Dahl/Rapp Rats
ated S rat colony are indeed S rats. Even then, because
only the present genetic contamination can be reliably
detected by the genetic markers described above, researchers still need to convince themselves that these
strains are indeed genetically purebred and free from
other episodes of contamination.
Acknowledgments
Portions of this work were supported by an American Heart
Association Grant-In-Aid and Harlan Sprague Dawley, Inc.
Dr Lewis is supported by Ginical Investigator Development
Award 1 K08 HL-02856-01 from the National Institutes of
Health, Bethesda, Md. Dr Warnock is supported in part by the
VA Research Service. We wish to thank Kimberly Vines for
her technical assistance with this project.
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J L Lewis, R J Russell and D G Warnock
Hypertension. 1994;24:255-259
doi: 10.1161/01.HYP.24.3.255
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