Hypertension: Methods and Protocols (Methods in Molecular Medicine)

501 Pages·2004·9.301 MB·English

by  Jerome P. Fennell, Andrew H. Baker

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Congenic/Consomic Models of Hypertension 3
1
Congenic/Consomic Models of Hypertension
Delyth Graham, Martin W. McBride, Nicholas J. R. Brain,
and Anna F. Dominiczak
Summary
Human essential hypertension is a complex, multifactorial, quantitative trait under poly-
genic control. Despite major recent advances in genome sequencing and statistical tools, the
genetic dissection of essential hypertension still provides a formidable challenge. Genetic mod-
els of essential hypertension such as the spontaneously hypertensive stroke-prone rat (SHRSP)
provide the scientist with genetic homogeneity, not possible within a human population, to aid
the search for causative genes. The principal strategy in the rat has been the identification of
quantitative trait loci (QTL) responsible for blood-pressure regulation by genome-wide scan-
ning. In this chapter we focus on congenic and consomic breeding strategies for the confirma-
tion of QTL and the genetic dissection of the implicated regions.
Key Words:Genotyping; polymerase chain reaction; microsatellite markers; quantitative
trait locus (QTL); congenic; consomic, hypertension; stroke-prone spontaneously hypertensive
rat (SHRSP); Wistar Kyoto rat (WKY).
1. Introduction
An important approach to the study of multifactorial, polygenic diseases
such as essential hypertension is the use of appropriate inbred animal models.
Major advantages of inbred animal models include genetic homogeneity and
the complete control of environmental influences. Most important, the ability
to produce specific intercrosses between normotensive and hypertensive strains
represents a powerful experimental tool for performing linkage analyses far
beyond the scope of human studies (1).
Many hypertensive rat strains have been produced over the past 30 yr, rang-
ing from the spontaneously hypertensive rat (SHR) and stroke-prone spontane-
ously hypertensive rat (SHRSP) to strains in which high dietary salt is
necessary to induce hypertension, such as the Dahl salt-sensitive and Sabra
From:Methods in Molecular Medicine, Vol. 108: Hypertension: Methods and Protocols
Edited by: J. P. Fennell and A. H. Baker © Humana Press Inc., Totowa, NJ
3
4 Graham et al.
rats. These models also exhibit end-organ damage phenotypes similar to those
seen in human essential hypertension, including left ventricular hypertrophy,
stroke, and renal failure (2–4). Most inbred rat strains have been derived from
Wistar-related stocks and others from Sprague-Dawley; however, as these
strains have a common origin, there is relatively little genetic diversity. The
exception is the fawn hooded rat, which may have a more distant origin (5).
The major strategy in the search for causative genes underlying complex
polygenic traits such as hypertension has been the identification of quantitative
trait loci (QTL) in F2 segregating populations (i.e., the genome-wide scan or
linkage analysis) (6). A QTL defines a large chromosomal region (approx 20–
30 cM) containing one or more loci controlling a quantitative trait. Numerous
linkage analysis studies in experimental crosses between hypertensive and nor-
motensive reference rat strains have been carried out over the past 10 yr, which
have identified QTL for blood pressure on every rat chromosome (6,7). Since
rodent QTL regions are too large to test functionally all putative candidate
genes, the identification of a blood pressure QTL can be considered as only
the first step toward causative gene identification (8). Further strategies include
production of consomic or congenic strains and substrains to confirm the exist-
ence of the QTL and to allow genetic dissection of the implicated region (9).
A congenic/consomic strain is one in which part of the genome of one rat
strain is selectively transferred to another by backcrossing a donor rat strain to
a recipient strain with appropriate selection. In the case of consomic strains
(chromosome substitution), a whole chromosome is transferred (10,11),
whereas in the case of congenic strain, a defined chromosomal segment (the
differential segment) is transferred. It should be noted that congenic strains
contain not only the selected differential locus, but also an associated length of
the surrounding donor chromosome. Consomic strains have proved to be
invaluable in the study of the nonrecombining Y chromosome effect on hyper-
tension(11–13). Furthermore, a large panel of consomic rat strains has recently
been developed (14), in which each autosome has been individually replaced.
Consomic panels can be used to determine the chromosomal location of genes
contributing to particular phenotypes and to generate congenic strains for posi-
tional cloning rapidly.
Classically, the congenic strain breeding procedure involves serially back-
crossing the donor strain to the recipient, while selecting progeny carrying the
desired QTL region in each generation of backcrossing (Fig. 1). According to
Mendelian laws, between 8 and 10 backcrosses are required to dilute the donor
genome into >99% that of the recipient. Brother–sister mating can then “fix”
the desired introgressed segment as homozygous and the congenic strain can
then be phenotyped. A variety of rat congenic strains has been produced in this
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Fig. 1. Construction of reciprocal congenic strains using SHRSP and WKY parental strains. Cross 1, WKY chromo-
somal regions introgressed into SHRSP background (strains A and B). Cross 2, SHRSP chromosomal regions introgressed
into WKY background (strains C and D).
5
6 Graham et al.
manner. In order to ensure transfer of entire QTL, original congenic strains
tend to have large chromosomal regions introgressed, therefore smaller
substrains (minimal congenics) are required to reduce the size and hence the
implicated region. In some cases there may be more than one QTL present in
any given region, therefore, several substrains are necessary to dissect the
implicated region (15). Substrains are constructed by crossing the original
congenic strain with the recipient stain and brother–sister mating the resultant
heterozygous offspring. Recombination events occurring at meiosis allow for
the production of progeny with smaller introgressed regions that can be selected
by genotyping densely spaced polymorphic markers (16).
The traditional breeding strategy takes approx 3–4 yr to produce a congenic
strain (17). This time can be shortened by utilizing a marker-assisted “speed”
congenic breeding strategy, previously tested in the mouse (18). Jeffs et al.
(19) were the first to show that this strategy can also be successful in the rat.
Lander and Schork (20)proposed that screening of polymorphic genetic mark-
ers covering the entire background of the genome could be used to select male
offspring with least donor alleles in their background. In this way, the next
round of breeding using the “best” male could dramatically reduce the time
taken to clear the background of the recipient strain (Fig. 2). It has been calcu-
lated that by four backcross generations, donor genome contamination would
be less than 1% if 60 background markers, spaced on average 25 cM apart,
were used for screening 16 males at each generation. This speed congenic ap-
proach therefore reduces the average time required to produce strains to approx
2 yr (19).
In this chapter we describe the methodology for a speed congenic/consomic
breeding strategy and high-throughput fluorescent genotyping using the stroke-
prone spontaneously hypertensive rat (SHRSP) and the normotensive Wistar-
Kyoto (WKY) strain.
2. Materials
2.1. Genomic DNA Isolation (Animal Tail Tip)
1. 0.5 M EDTA (pH 8.0): Sterilization by autoclaving.
2. Nuclei lysis solution: (Promega, cat. no. A7943).
3. Protein precipitation solution: (Promega, cat. no. A7953).
4. 20 mg/mL Proteinase K.
5. Isopropanol.
6. 70% Ethanol.
7. Benchtop microcentrifuge.
8. Whirli mixer (e.g., FSA Laboratory Supplies)
9. Hybridization oven.
10. Sterile pipet tips.
Congenic/Consomic Models of Hypertension 7
Fig. 2. Congenic strain construction, illustrating differences between the traditional
and marker-assisted speed congenic approach. The arrows indicate the backcross at
which background heterozygosity is theoretically the same. Decreasing shades of gray
to white represent the serial dilution of the donor genome in the genetic background.
D, donor strain alleles; R, recipient strain alleles; B, backcross; F1, first filial generation.
8 Graham et al.
2.2. Quantification of Genomic DNA
1. UV/visible Spectrophotometer (e.g., Pharmacia Biotech Ultrospec 2000).
2. Quartz cuvets.
3. Parafilm.
2.3. Polymerase Chain Reaction
1. Deep 96-well plate.
2. Thermo-fast 96-well skirted polymerase chain reaction (PCR) plates (ABgene
cat. no. AB-0800).
3. PCR plate with adhesive cover.
4. 1 mM dNTPs stocks.
5. 10X Hotstar Taq DNA polymerase buffer (Qiagen, UK) (seeNote 1).
6. Forward and reverse primers (20 pmol/µL conc.).
7. 0.5 U/µL Hotstar Taq DNA polymerase (Qiagen, UK).
8. Thermal Cycler hot lid PCR machine (e.g., MJ Research PTC-225 Peltier).
9. Multichannel electric pipet.
2.4. PCR Pooling
1. Thermo-fast 96-well skirted PCR plates (ABgene cat. no. AB-0800).
2. Multichannel electric pipet.
2.5. Casting Gels for the ABI Prism 377XL
1. Powder-free gloves (seeNote 2).
2. 50% Long Ranger gel solution (Cambrex Corporation, NJ).
3. Ultrapure urea.
4. Bio-Rad AG 501-X8 (D) deionizing resin.
5. Deionized water.
6. 50-mL Measuring cylinder.
7. 10% w/v Ammonium persulfate.
8. Whatman cellulose filters.
9. 10X TBE: 0.89 M Tris-borate pH 8.3, 20 mM Na EDTA.
2
10. TEMED (N,N,N',N'- tetramethylehtylenediamine, Sigma 7024).
11. 60-mL syringe.
12. Bubble catcher.
13. Bulldog clips.
3. Methods
3.1. Marker-Assisted “Speed” Congenic Strategy
1. Congenic strains are constructed using a speed congenic (or marker-assisted)
strategy whereby various segments of the rat chromosome of interest are
introgressed from the normotensive WKY strain to the genetic background of the
SHRSP, and in the reciprocal direction from SHRSP to the genetic background
of the WKY.
Congenic/Consomic Models of Hypertension 9
2. Reciprocal F1 generations are produced by crossing WKY and SHRSP (one
breeding pair per cross is sufficient). Resulting male F1 hybrids are mated to the
desired recipient strain (WKY or SHRSP) females (seeNote 3).
3. Microsatellite markers throughout the chromosomal region of interest, and addi-
tional markers broadly spanning the remaining genome, are genotyped (seeSub-
heading 3.3.) in the offspring from this first backcross (seeNote 4). Those males
identified as heterozygous for marker alleles within the chromosomal region of
interest, but with most homozygosity for recipient alleles throughout the remain-
ing genome, are selected as “best” males for breeding (see Note 5). These best
males are mated with recipient strain females to produce a second backcross gen-
eration and the offspring are genotyped.
4. Procedure is repeated by backcrossing “best” male offspring until all donor alle-
les in the genetic background (indicated by the background markers) are eradi-
cated (seeNote 6). Approximately four to five backcross generations are required
to achieve recipient strain homozygosity in all background markers. The differ-
ential chromosomal region is then fixed and made homozygous by crossing ap-
propriate males and females.
5. Fixed congenic strains are maintained by brother–sister mating.
6. Confirmation of successful QTL capture by phenotypic measurement within a
congenic strain is followed by congenic substrain (or minimal congenic) produc-
tion with smaller donor regions. This strategy dissects the introgressed region,
allowing further localization of the QTL.
7. Congenic substrain production is undertaken by backcrossing congenic males to
recipient females to yield rats heterozygous within the original introgressed seg-
ment, while maintaining recipient strain homozygosity in the remaining genome.
8. Resulting heterozygous F1 rats are intercrossed and the offspring genotyped to
identify appropriate males and females with smaller regions of donor allele
homozygosity. These smaller regions are fixed and the new substrains maintained
by brother-sister mating.
9. Linked databases for management of genotyping data and breeding protocols
were designed and implemented in-house (Microsoft Access 2000).
3.2. Marker-Assisted “Speed” Consomic Strategy
An identical speed breeding strategy can be applied to consomic strain pro-
duction. In this case the entire chromosome of interest is introgressed from
donor into the recipient strain. For production of Y-chromosome consomic
strains, slight modifications to the breeding strategy are required, as follows:
1. The Y chromosome contains a large nonrecombining region; therefore the origin
of the male F1 progenitor will determine the Y chromosome in all resulting F1
hybrid males.
2. Microsatellite markers broadly spanning all of the autosomes are genotyped (see
Subheading 3.3.) in the offspring from a first backcross generation. “Best” males
for backcrossing to recipient strain females are chosen on the basis of least het-
erozygosity throughout the autosomal background.
10 Graham et al.
3. Backcrossing is continued until all donor alleles in the genetic background (indi-
cated by background markers) are eradicated.
4. Y-chromosome consomic strains can be fixed and maintained by backcrossing to
recipient strain females.
3.3. High-Throughput Fluorescent Genotyping
3.3.1. Genomic DNA Isolation From Animal Tail Tip
1. Briefly anaesthetize the animal with halothane/oxygen, remove a 4-mm tip from
the tail, and immediately cauterize wound (seeNote 7).
2. Prepare the tail tip samples for digestion; add 120 µL of 0.5 MEDTA to 500 µL
of nuclei lysis solution for each sample into an appropriate-sized tube.
3. Chill the mix on ice for 5 min.
4. Add 600 µL of the prepared mix to each tail tip sample and add 17.5 µL of 20 mg/
mL proteinase K solution. Incubate the samples in a rotating hybridization oven
at 37°C overnight (seeNote 8).
5. Allow the samples to cool to room temperature.
6. Add 200 µL of protein precipitation solution to each of the samples and mix by
vortex for 20 s.
7. Chill the samples on ice for 5 min.
8. Centrifuge the samples for 4 min at 13,000g(full speed in Eppendorf centrifuge)
at room temperature (seeNote 9).
9. Transfer the supernatant to a fresh 1.5-mL Eppendorf.
10. Add 600 µL of room-temperature isopropanol and mix by inversion (seeNote 10).
11. Centrifuge the sample for 2 min at 13,000g at room temperature.
12. Carefully decant the supernatant and ensure that there is a white pellet visible and
it is not disturbed.
13. Wash the DNA pellet by adding 1 mL of 70% ethanol.
14. Centrifuge the sample for 1 min at 13,000g at room temperature.
15. Carefully remove the supernatant by pipet and invert the tube on a piece of absor-
bent paper. DNA pellet and then store the samples in a 4°C fridge overnight.
3.3.2. Quantification of Genomic DNA
1. Allow the spectrophotometer to self-calibrate.
2. Blank-correct by adding 1 mL of sterile water to a clean quartz cuvet and insert it
into the main reading holder.
3. Replace the cuvet in the main reading holder. The absorbance reading should be
zero.
4. Empty the cuvet and wash thoroughly with distilled water.
5. Add 995 µL of sterile water to the cuvet.
6. Add 5 µL of sample DNA to the cuvet and mix well by inversion (cover the cuvet
opening with parafilm and mix). This reading will give a reading for the 1/200
dilution factor for the DNA.
7. Insert the cuvet into the main reading holder and press Run.
Congenic/Consomic Models of Hypertension 11
8. Record the OD concentration value given on the machine.
260
9. To obtain the 260/280 ratio value, press the down arrow key (seeNote 11).
10. Add a further 5 µL of the sample DNA to the cuvet (mix as before) and repeat as
before. This value will give a reading for the 1/100 dilution factor for the DNA.
11. Empty the contents and wash the cuvet with distilled water and repeat the proce-
dure for each sample.
12. Make DNA stocks of 20 ng/µL.
3.3.3. Polymorphic Fluorescent Microsatellite Markers
Polymorphic microsatellite markers are synthesized with a fluorescent mol-
ecule (either TET, FAM, and HEX available from ABI) at the 5' end of the
forward primer. Microsatellite markers of a similar size are synthesized with
different color fluorophores. This allows greatest flexibility when considering
pooling strategies (seeSubheading 3.3.5.).
3.3.4. Polymerase Chain Reaction
1. Prepare deep-well plate of 20 ng/µL DNA stock: Add the calculated volume of
DNA to a sterile deep-well plate and make the volume up to a total of 500 µL
with sterile water. This will provide the template DNA for future PCR procedures.
2. Label thermo-fast 96-well plates with the appropriate primer used for PCR
and date.
3. Transfer 5 µL of the previously prepared 20-ng/µL DNA template with a multi-
channel pipet.
4. Prepare a PCR master mix (seeNote 12). Per sample:
a. 2.0 µL 10X Hotstar Taq buffer.
b. 4.0 µL dNTPs (1-mM stock).
c. 0.5 µL each of forward and reverse primer (20 pmol/µL stock).
d. 6.96 µL sterile water.
e. 0.04 µL Hotstar Taq enzyme (seeNote 13).
5. Add 15 µL of the prepared master mix to the samples in each well.
6. Firmly place an adhesive cover on the prepared PCR plate and seal tightly.
7. Place the plate in a PCR machine and tighten the lid (seeNote 14).
8. Standard themocycler PCR conditions:
a. 95°C for 15 min (1X).
b. 95°C for 1 min.
c. 55°C 1 min.
d. 72°C for 2 min (35X).
3.3.5. PCR Pooling
Pooling of PCR products before loading samples on to the acrylamide gel
reduces the number of lanes required for genotyping and is a key step for high-
throughput genotyping. Pooling involves mixing between 5 and 10 PCR
amplicons from a number of microsatellite amplification reactions into a single