Chromosome 2 Reciprocal Congenic strains to evaluate the effect of the genetic background on blood pressure
SMJ 2002:47(1): 7-9
F.J. Carr, C.D. Negrin, J.S. Clark, D Graham, M.W. McBride, N.W. Anderson, A.F. Dominiczak
Department of Medicine and Therapeutics, University of Glasgow, Western Infirmary, Glasgow.
Abstract: The localisation of quantitative trait loci (QTL) is the first step towards gene identification. This is then verified by the construction of reciprocal congenic strains. The hypertensive SHRSP and normotensive WKY strains were used in a speed congenic approach to confirm the existence of a QTL on rat chromosome 2. Systolic baseline and salt loaded blood pressures were measured by radiotelemetry. Transfer of the chromosome 2 blood pressure QTL region from WKY into an SHRSP background significantly reduced blood pressure, with the increased significance at the salt loaded period, compared to the SHRSP. The reciprocal congenic blood pressure showed a significantly increased baseline systolic pressure compared to the WKY, with no change in significance at the salt loaded period. Thus we have successfully captured a gene(s) which contribute to blood pressure regulation in both congenic strains. This will facilitate further positional cloning of the causative genes first in this model and then in human essential hypertension.
Keywords: Hypertension, genes, congenic strains
Introduction
Human essential hypertension is a complex, multifactorial, polygenic trait and which is difficult to study without the use of a suitable animal model. The spontaneously hypertensive stroke prone rat (SHRSP) is one such animal model which is found to be both stroke prone and hypertensive when compared to the normotensive reference strain Wistar-Kyoto rats (WKY) and can therefore be used to study the genetics of hypertension. The localisation of a quantitative trait locus (QTL) using a total genome scan is the first step towards gene identification. The term QTL is used to describe a chromosomal segment that may harbour one or more loci controlling a quantitative trait such as blood pressure.This approach for mapping genetic loci in the rat which contributed to blood pressure regulation was pioneered by Hilbert et al. in 1991.
1 Linkage studies previously undertaken by our group2 revealed two separate QTLs for blood pressure on rat chromosome 2. The existence of a QTL on rat chromosome 2 has also been confirmed by Deng et al. in 1997.3 To identify the genes responsible for hypertension the construction of congenic rat strains is the next essential step.A congenic strain is traditionally constructed by the serial backcrossing of a donor strain with a recipient strain. Chromosomal regions are monitored at each backcross for heterozygosity using polymorphic microsatellite markers. After eight to twelve backcrosses, according to Mendelian laws, more than 99% of the genetic background is that of the recipient strain. On completion of the backcrossing, the final step of brother x sister mating fixes the strain so that the desired chromosomal region is homologous for the donor’s alleles in one quarter of the offspring.
Congenic strain production can be made quicker by screening polymorphic marker loci distributed throughout the genome. This allows for the selection of a ‘best male’ at each stage of backcrossing which has the least amount of donor alleles in the genetic background whilst still maintaining heterozygosity at the chromosomal region of interest. This marker-assisted or "speed" congenic strategy was previously used in mice.
4 Our group initiated its use in the rat by successfully breeding several congenic strains.5 By selecting for the ‘best male’ the number of backcross generations needed to establish congenic strains in the rat is reduced to approximately five instead of the traditional eight to 12. This speed congenic strategy can save nearly two years of backcrossing at the price of genotyping hundreds of markers.Methods
This speed congenic approach was used to produce congenic strains of contrasting genetic backgrounds, that is reciprocal congenic strains. The hypertensive SHRSP
Gla strain and the normotensive WXYGla strain were used to produce reciprocal congenic strains to evaluate the effect of the genetic background on blood pressure. In the reciprocal strains the region introgressed was identical to avoid confounding effects of additional QTLs. Two reciprocal control congenic strains were also produced to determine the presence of potentially artifactual ‘passenger’ loci, which could have a confounding effect on blood pressure.The rats were phenotyped via a radiotelemetry method. Surgical implantation of a telemetry transmitter into the abdominal aorta was undertaken when animals were 12 weeks of age. The rats were given one week to recover after anaesthesia followed by measurement of a five week baseline period ending in a three week salt loaded period during which the rats received 1% NaCl in their drinking water. Measurements were made for 10 seconds every five minutes to produce a total of 10,080 measurements during the baseline period and 6,048 during the salt loaded period. These blood pressure readings were averaged for each hour and standard error of the mean was calculated. Comparisons of congenic strains with their progenitor strain were undertaken with repeated measures ANOVA, p values <0.05 were considered to be significant.
To obtain DNA from animals to genotype a 4mm tip from the tail was removed from offspring under anaesthetic at four weeks of age prior to radiotelemetry. DNA was extracted and genotyping was performed using polymerase chain reaction
amplification of DNA around the 83 polymorphic microsatellite markers from total genomic DNA with the use of specific primer pairs obtained from Research Genetics (Alabama, USA) or Sigma Genosys Biotechnology (Cambridge, UK).Results
The congenic strains were produced (Fig 1), phenotyped and genotyped. In the nomenclature of the congenic strains the first abbreviation refers to the recipient background strain and the second refers to the donor strain which is the genotype of the introgressed region. The number 2 refers to rat chromosome 2 and a and b are arbitrarily assigned to each strain.Transfer of the region of rat chromosome 2 from the WKY into an SHRSP background significantly lowered both baseline
and salt loaded systolic blood pressures by approximately 20 and 40mmHg, respectively in male congenic rats (n=6) when compared to the SHRSP parental strain (n=6) during the night time period (F=9.9, p=0.016; and F=15.5, p=0.003, respectively)(Fig 2).In contrast, transfer of an identical region from the SHRSP into a WKY background significantly increased night time systolic blood pressures by approximately 20mmHg in male congenic rats (n=6) compared to the WKY progenitor strain (n=6) during the baseline period. (F=21.7, p=0.0009). However, with the WKY normotensive strain as the background strain there was no further increase in systolic blood pressures during the salt loaded period (F=24.4, p=0.0006)(Fig 3). The reciprocal control congenic strains showed no deviation from the blood pressures recorded in their respective parental strain.(Figs 2 and 3).
Summary and conclusions
These results confirm the existence of a QTL on chromosome 2 in the rat and also the viability of the speed congenic approach. Transfer of the entire region of rat chromosome 2 containing both QTLs from a WKYGla into a SHRSPGla genetic background significantly lowered both baseline and salt loaded systolic blood pressures by approximately 20mmHg and 40mmHg, respectively, as determined through continuous and direct recording with radiotelemetry. In the reciprocal strain, transfer of the same region of chromosome 2 from SHRSPG1a into a WKYGla genetic background significantly increased both baseline and salt loaded systolic blood pressure by approximately 20mmHg.
These results provide evidence that the genetic background of a given congenic strain has an effect on the phenotype. It can also be seen that the SHRSP background is permissive for a ‘salt effect’; that is an exaggeration in the blood pressure when on 1% salt whilst this effect is not observed with the WKY background.
Acknowledgement: This extended abstract was presented at the Glasgow meeting of the Scottish Society of Experimental Medicine on the 26 of May 2000 and Fiona Carr was awarded the Sir James Black award for the best oral presentation. This work is funded by the British Heart Foundation Programme and Project Grants to AFD