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. 2003 Nov 14;554(Pt 1):46–55. doi: 10.1113/jphysiol.2003.052613

Application of chromosomal substitution techniques in gene-function discovery

Allen W Cowley Jr 1, Richard J Roman 1, Howard J Jacob 1
PMCID: PMC1664739  PMID: 14678490

Abstract

A consomic rat strain is one in which an entire chromosome is introgressed into the isogenic background of another inbred strain using marker assisted selection. The development and initial physiologic screening of two inbred consomic rat panels on two genetic backgrounds (44 strains) is well underway. The primary uses of consomic strains are: (1) to assign traits and quantitative trait loci (QTL) to chromosomes by surveying the panel of strains with substituted chromosomes; (2) to rapidly develop congenic strains over a narrow region using several approaches described in this review and perform F2 linkage studies to positionally locate QTL in a fixed genetic background. In addition, consomic strains overcome many of the problems encountered with segregating crosses where, even if linkage is found, each individual in the cross is genetically unique and the combination of genes cannot be reproduced or studied in detail. Consomic strains provide greater statistical power to detect linkage than traditional F2 crosses because of their fixed genetic backgrounds, and can produce sufficient numbers of genetically identical rats to validate the relationship between a trait and a particular chromosome. These strains allow studies to be performed in a replicative or longitudinal manner to elucidate in greater detail the sequential changes responsible for the observed phenotypes of these animals, and they enable one to assess the impact of a causal gene region in a genome by allowing comparisons of the effect of replacement of a specific chromosome upon a disease susceptible or resistant genomic background. Consomics can be used to quickly develop multiple chromosome substitution models to investigate gene–gene interactions of complex traits or diseases. Finally, they often provide the best available inbred control strain for particular physiological comparisons with the inbred parental strains. Consomic rat strains are proving to be a unique scientific resource that greatly extends our understanding of genes and complex normal and pathological function.

The need to relate the complete set of sequenced genes to pathways of biological function now represents one of the great challenges of modern biology. It is evident from the data posted in various public genome databases such as MGD, RGD, LocusLink and OMIM that there remain at least 15–20 000 genes that have no confirmed action in rat, mouse, man or other mammalian organisms. The function for the majority of the other genes has been inferred based on sequence homology with cDNAs and expressed sequence tags (ESTs) reported in other organisms and will eventually require confirmation in one's organism(s) of interest (Lander et al. 2001; Nadeau et al. 2001; Venter et al. 2001). The goal of this brief review is to present an overview of how techniques of chromosomal substitution (either a part of chromosome called a congenic strain, or the entire chromosome called a consomic strain) are being used to help decipher the relationships between genes and complex biological functions.

As described in another part of this thematic series (McBride et al. 2004), genetic linkage studies were first used to determine the chromosomal locations of both simple and complex traits. We have used segregating crosses of rats to develop an extensive genetic map of cardiovascular function (Stoll et al. 2001; Moreno et al. 2003) and identified more than 90 interesting quantitative trait loci (QTL) that mapped to 19 chromosomes using this approach. There are, however, a number of limitations to the linkage analysis approach. Besides being slow and labourious, large populations are required to achieve the statistical power necessary for defining QTL. Importantly, even in the most tightly controlled intercross study, genetic background differences among the individual F2 rats can modify or mask the effects of genetic differences, thereby reducing the power of the analysis. To overcome these and other limitations described below, chromosomal substitution approaches were developed in mice as described by Nadeau et al. (2000). For our own interests, the rat was chosen as the model system because of the importance of rat model systems in diseases such as hypertension, diabetes, and renal failure, in which many different rat models have been extensively characterized. Presently there are 234 different inbred strains of rats that have been developed over the past 80 years for the study of many complex human diseases (Colle et al. 1983; Mordes et al. 1987; Gill et al. 1989; Kwitek-Black & Jacob, 2001; Jacob & Kwitek, 2002). Description of these strains can be found at the rat genome database website (http://www.rgd.mcw.edu). Because of these many inbred rat strains, and given the extensive physiological knowledge available for the rat, we have been developing genomic and physiological approaches to speed our understanding of genes and function in rats. The chromosomal substitution techniques represent one of these important tools (Roman et al. 2002; Cowley, 2003). The manner in which these model systems are developed and their applications in gene-function identification is reviewed below. Since congenic strains were initially developed for confirmation and further localization of QTL that were identified in linkage studies, we first explain how such congenic strains were originally developed.

Congenic rat strains

Chromosomal substitution techniques were initially developed to confirm the existence of a QTL in a chromosomal region that has been identified by linkage analysis. They were first used in ways that substituted only small portions of an individual chromosome containing the segregating phenotype of interest (such as blood pressure) from a control strain into a susceptible strain to determine if this region of the chromosome would alter the trait of interest (or vice versa). The functional importance of a genomic region could thereby be validated. The substitution of a narrow region within a chromosome into the isogenic background of another inbred strain is referred to as a ‘congenic’ strain. This approach was pioneered by Snell (1948) in his Nobel work on the major histocompatibility complex (MHC). Typically, congenics are generated in 10–12 generations of backcrossing (Falconer, 1989). As an example, two inbred strains of rats in which all of the alleles are homozygote are intercrossed to produce an F1 generation as shown in Fig. 1. A normotensive Brown Norway (BN) rat is intercrossed with a Dahl S rat (salt-sensitive strain of hypertensive rat, SS). The alleles on every chromosome of the F1 generation rats are heterozygous, having received one haploid strand of each chromosome from the parental rat. Rats of the F1 generation are then backcrossed to the recipient genomic background of the parental SS strain (in this example). Within the F1 animals, meiotic recombination events between the two parental chromosomes result in 40–50% of the chromosomes being passed on to the next generation being recombinant (mixed) (Nadeau et al. 2000). The goal is to then carry out a series of backcrosses with the parental strains to wash away the donor genome with the exception of the region of interest. To accomplish this requires 10 generations of backcross breeding with some type of marker or phenotype being used to confirm that the region of interest has been moved. If one wishes to substitute a small part of chromosome 1 from the BN strain into the SS strain (Fig. 1A), one would select a N2 backcross rat that was heterozygous (+/–) within the region of interest and backcross this rat with the inbred homozygous parental SS strain. This backcross breeding process to fix the genetic background of the congenic strain can be accelerated by genotyping of the entire genome (∼100 markers) and selecting the breeder(s) based on several characteristics. This is determined by carrying out total genome scans. First, the rats must be heterozygous within the region of interest. Second, rats are selected that carry a preponderance of homozygous +/+ Dahl S (recipient) alleles throughout the remainder of the genome. This process of genotyping carried out with about 100–200 polymorphic microsatellite markers is referred to as ‘marker-assisted selection’ (Lander & Thompson, 1990) or ‘speed congenics’ (Markel et al. 1997) since it greatly speeds the process of selecting the rats for optimum backcrossing to the parental strains in order to achieve a homozygote state for all alleles except those on the target chromosome. Mendelian genetics predicts that, by chance, each backcross would reduce heterozygosity of each marker or allele by 50%, in an exponential decay model. Marker-assisted selection with whole genome scans reduces the time required for this process by nearly one-half. Therefore, a process that could require 4–5 years can be reduced to 2–3 years with marker-assisted selection. These techniques are in routine use to generate both congenic mice (Markel et al. 1997) and rat (Jeffs et al. 2000) strains.

Figure 1. Schematic representation of the generation of a congenic strain from two genetically different rat strains.

Figure 1

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A, parental strains Brown Norway (BN) and Dahl salt-sensitive (SS) are intercrossed for the generation of a heterozygous F1 population. The F1 is then crossed with the parental background of interest (in this example, the SS) to generate an N2 population. The N2 rats are then backcrossed 6–10 generations using marker-assisted selection of offspring, in order to substitute a selected genomic region from the BN rat. B, a male and female rat, selected by genotyping for this specific target region containing the phenotype of interest, are then mated. 25% of the offspring from this cross will be homozygotes for this region. These rats are then inbred to produce a stable inbred congenic strain.

The final cross involves intercrossing a male and female that are heterozygous SS/BN for markers within the region of interest (Fig. 1B), and homozygous for the SS parental strain on all other chromosomes. Approximately 25% of the progeny of this intercross will be homozygous BN/BN in this region while the alleles of all other chromosomes will be homozygous SS/SS (isogenic SS). These congenic rats can then be brother–sister mated to produce and maintain a colony of inbred congenic rats. Congenic rats and mice strains have been used to successfully narrow QTL regions of interest (Garrett et al. 2002, 2003a,b; Meng et al. 2003) in an effort to identify candidate genes of hypertension, but this approach has proven to be slow and inefficient for traits with many QTL. For this reason, an alternative strategy was developed to speed the process, the development of consomic panels.

Male rats of the consomic strains that were created using our current breeding strategy harbour the Y chromosome from the SS strain. However, we also created a consomic strain in which the Y chromosome of the BN stain was transferred into the autosomal background of the SS rat. This was done by crossing a male BN rat with a female SS to create the F1 generation. Thereafter, male rats were backcrossed with female SS rats for several generations until the SS background was fixed across the genome. This SS-YBN consomic strain can be used to explore the influence of the Y chromosome on various phenotypes in SS rats. This strain can also be used to switch the Y chromosome in any of our consomic strains from SS to BN in two generations by mating a male SS-YBN consomic rat with a female rat from any of the SS-BN consomic strains. Male rats of such a cross carry the Y chromosome from BN rats and are heterozygous on the consomic chromosome. By backcrossing male F1 rats with female rats of the original consomic strain one can create and select males that carry the Y chromosome of BN rats and are again homozygous for the BN alleles on the autosomal chromosome of interest. This new strain can be used to examine the interactions between the Y chromosome and the chromosome of interest on the expression of various genes and phenotypes.

Even mitochondrial DNA (the ‘neglected chromosome’) can be substituted between strains. Mitochondrial DNA is inherited from females. Because we always backcrossed male rats to female SS rats the consomic strains we created carry the mitochondrial genome of SS rats. However, one could easily create another consomic strain carrying the mitochondrial genome of BN rats by crossing a male SS rat with a female BN rat. Female F1 rats, and rats of all subsequent generations, would be backcrossed with male SS rats for several generations until the SS background was fixed across the genome. The resultant consomic strain would be homozygous for the SS autosomes but would carry the mitochondrial genome of BN rats. Again this strain could be used to switch the mitochondrial genome in any of our consomic strains by crossing male rats of the consomic strain of interest with a female rat of the consomic strain carrying the mitochondrial genome of the BN strain. The transfer could be completed by backcrossing male rats of this cross with female rats with the BN mitochondria and selecting rats that are homozygous for the BN alleles on the autosomal chromosome of interest but harbouring the mitochondrial genome of BN rats.

Consomic rat panels

It is clearly not practical to build a complete series of narrowly overlapping congenics for all the cardiovascular QTL across the genome using the approach described above. However, such an approach is feasible if the length of the chromosomal regions are very large. For example, if one transfers whole chromosomes, then only 22 rat strains (20 autosomes plus the X and Y) need to be maintained to confirm QTL throughout the genome.

Figure 2A illustrates the strategy of creating a consomic strain of rats. The principle is the same as creating a congenic strain, except an entire chromosome is transferred. An F1 generation rat is backcrossed to the parental strain (SS in this example) using marker-assisted selection. The alleles on the target chromosome for substitution are selected to be heterozygous through 4–8 backcrosses and the rats that exhibit the greatest homozygosity for the SS parental strain are selected for backcrossing. By the 8th generation, all chromosomes except the target chromosome become fixed for the parental strain. As shown in Fig. 2B, a male and female rat that are homozygous for the SS alleles on all chromosomes except the target chromosome on which they are heterozygous are then intercrossed with the resulting approximately 25% (depends on the size of the chromosome) of the offspring becoming homozygous BN/BN (consomic) for the target chromosome. These rats can then be inbred for a renewable resource of consomic rats.

Figure 2. Schematic representation of the derivation of a consomic strain.

Figure 2

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A, similar to the congenic strain development, parental strains are intercrossed and generate a heterozygous F1 population, which is backcrossed with the parental SS strain to get the desired background. The N2 rats that are determined by genotyping to be heterozygous along the target chromosome, are then backcrossed for 4–8 generations to yield offspring with an isogenic SS background for all but the target chromosome. B, selected rats are then brother–sister mated and 25% of the offspring will be homozygotes for the chromosome of interest. These rats are then inbred to produce a stable inbred consomic strain.

It is important to remember that consomic rats are inbred strains and alleles are homozygous at all loci. Maternal and paternal chromosomes are identical so these inbred strains are essentially genetic ‘twins’ and multiple crosses between two strains can be set up without complications of introducing modifier genes. Consomic inbred rat strains thereby solve one of the greatest problems found in studying human populations, that of heterogeneity, while allowing the production of a large number of progeny. This production level is essential for detailed physiological studies, each rat then adding knowledge about the same strain.

Two panels, 22 consomic strains in each panel (44 strains) are being developed in which chromosomes from a normotensive BN rat are each substituted (one at a time) into the genetic background of the SS/JrHsdMcwi Dahl salt-sensitive (SS) or FHH/EurMcwi Fawn Hooded Hypertensive rats (FHH). These strains are available for purchase from Charles River Laboratory (Wilmington, MA, USA). The SS rat is a commonly used model for salt-sensitive hypertension (Rapp, 1982; Zhou et al. 2000; Cosentino et al. 2002), insulin resistance (Buchanan et al. 1991; Kotchen et al. 1991; Zhang et al. 1994), hyperlipidaemia (Reaven et al. 1991), endothelial dysfunction (Luscher et al. 1987; Gauthier-Rein & Rusch, 1998; Boulanger, 1999; Quaschning et al. 2001), vascular injury (Takenaka et al. 1992; Boegehold, 2002), cardiac hypertrophy (Ganguli et al. 1979), and glomerulosclerosis (Roman & Kaldunski, 1991; Cowley & Roman, 1996; Cowley et al. 2000, 2001). The FHH rat is a model of systolic hypertension, renal disease, pulmonary hypertension, a platelet function bleeding disorder, alcoholism, and depression (Provoost et al. 2002; Datta et al. 2003).

Once a strain is available containing a whole substituted chromosome on an isogenic background (consomic strain), one can then develop a congenic strain over a narrow region in only two generations following an intercross with the consomic and parental strain. A consomic strain therefore has a full-length chromosome from one inbred strain introgressed into the isogenic background of another inbred strain. Figure 3 shows how consomic rats (SS-1–22BN) can be intercrossed with the inbred parental strain to produce a congenic strain. Rats of the backcross generation become heterozygous on the single target chromosome of interest as a result of the recombinations on the target chromosome while the other alleles on the other chromosomes remain homozygous at all locations (isogenic) thereby avoiding the need to backcross to wash away the donor genome. Male and female rats that are heterozygous within the same region of the targeted chromosome are then mated to obtain rats that are homozygous for the donor alleles at this site. The narrow region that modifies the trait of interest can then be rapidly determined. It should be recognized that this leaves some heterozygous sites on the ends of the congenic region, but once the phenotype of interest is found to be modified within the congenic region, the heterozygosity can be fixed to homozygosity by further rounds of backcross breeding. If a complete panel of consomic rats exists as a general resource, a congenic inbred rat strain can therefore quickly be generated within any chromosome for the positional cloning of genes as will be discussed in greater detail later in this review.

Figure 3. Generation of congenic rats from consomic strains.

Figure 3

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The parental strain is crossed with the consomic strain, to generate an F1 population with identical genetic background and a heterozygous target chromosome. These F1 rats are intercrossed to generate an F2 population of rats whose target chromosome will be congenic, due to recombination events. Two similar F2 rats are selected (by genotype) and mated to fix the region of interest.

A single consomic panel is limited in searching for causal genes related to a complex disease in that it remains a two allele system. For this reason, we chose to build two reciprocal consomic panels to provide a three allele system. A survey of 48 commonly used inbred strains of rats has revealed that on average there are six alleles (range 1–13 alleles) for ∼5000 genetic markers tested (Steen et al. 1999). The three strains that we have chosen to study (BN, SS and FHH) contain an average of three alleles, indicating that on average our consomic panels will capture 50% of the genetic variance present in the commonly used inbred strains of rats. Since the focus of our research programme is on diseases of the heart, vasculature, kidney, and the pulmonary system, these three strains were also chosen because they provided maximal differences in response to stressors of these systems. The common replacement of the BN genomic background also enables investigators to construct new models containing multiple substitutions to study gene–gene interactions by combining consomics and/or congenics with a common isogenic genomic background. It is possible that some genetic traits detected in traditional linkage studies may be lost if the expression of these traits is dependent upon cosegregation of several loci from multiple chromosomes. Such loci may be understood by constructing consomic or congenic models containing multiple substitutions. It may also be pointed out that that if one wishes to establish the patterns of inheritance of traits, inbred consomic strains provide only limited information in this regard. Although a consomic or congenic strain will reveal whether a trait is dominant or recessive, one cannot ascertain whether a trait is a co-dominant single allele phenotype (since both alleles of inbred congenics are homozygous).

The initial stimulus to generate consomic panels of rats was based on their use for rapid development of congenic model systems for gene identification by positional cloning. However, in the process of developing these panels, it was discovered that these consomic rats could provide many other important advantages. As discussed in the remainder of this review: (1) they can be used to map physiological function; (2) they enable one to assess the impact of a causal gene region in a permissive genome by allowing comparisons of the effect of replacement of a specific region in a disease susceptible genomic background; (3) they provide the best available inbred control strains for physiological studies; (4) since they are inbred strains, they provide a renewable animal resource; (5) they can be used to quickly develop other polygenic models to investigate gene–gene interactions of complex traits or diseases.

Identification of chromosomes of interest – mapping of physiological functions

Complex traits have posed special challenges for traditional linkage analysis studies. To map a complex trait such as blood pressure one must contend with gene–gene and gene–environment interactions, genetic heterogeneity, low penetrance, and limited statistical power. More systematic methods for genes underlying these complex traits have been needed as well as better standards of proof of gene discovery (Glazier et al. 2002). One of the most important uses of consomic rat panels is that they provide a greater power for mapping of traits and QTL where even weak effects can be identified in far fewer rats than are required with segregating crosses and other mapping methods (Nadeau et al. 2000). Chromosomal substitution strains can be used to validate QTL regions obtained by traditional linkage analysis studies. One of the most important uses of consomic stains, however, is that the availability of an entire panel of consomic rats enables the investigator to identify the chromosomes responsible for traits of interest. One need not even carry out genotyping to map such traits of interest given the availability of a consomic panel. For example, we have recently identified chromosomes harbouring genes that contribute to the development of hypertension-induced glomerulosclerosis in the SS rats using a consomic rat panel. It was known that SS rats rapidly develop severe hypertension, glomerular sclerosis, renal interstitial fibrosis and proteinuria following exposure to a high salt diet. Urinary protein excretion is a commonly used index of renal disease that can readily be determined with a 24 urine collection. After several weeks of a high salt intake (4–8% NaCl), SS rats become very proteinuric while BN rats exhibit no detectable change in protein excretion (Stoll et al. 2001). As seen in the partial consomic panel shown in Fig. 4, substitution of BN chromosome 13 or 18 into the isogenic background of the SS rats substantially reduced microalbuminuria and this was associated with pronounced improvement of the degree of glomerulosclerosis. Simply phenotyping these consomic rat strains enabled the identification of two chromosomes that are important in determining whether a high salt diet will lead to hypertension and renal disease.

Figure 4. Parental SS rats exhibit very high levels of mean arterial pressure (MAP) and urine albumin (microalbuminuria) compared to BN rats.

Figure 4

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Substitution of chromosome 13 from the BN rat into SS strains (SS.13BN) or chromosome 18 (SS.18BN), significantly reduced the severity of hypertension and microalbuminuria. This indicates the presence of important QTL on chromosomes 13 and 18 involved in blood pressure control and renal dysfunction. Rats were fed a high salt (8%) diet for 3 weeks prior to study. MAP is the average pressure measured across three days. Microalbumin was determined in urine collected on the third day of pressure recording. *P < 0.05 compared to SS.

We have previously mapped QTL in male rats for hypertension, salt-sensitivity, and proteinuria on chromosome 18 in SS rats (Cowley et al. 2000; Stoll et al. 2001). These QTL were verified by chromosomal substitution (Kaldunski et al. 2002). On the other hand, in this same linkage study, we did not identify any blood pressure or renal disease QTL on chromosome 13 (Stoll et al. 2001), despite a previous linkage study using SS rats that did map blood pressure QTL to chromosome 13 (Rapp et al. 1994; Rapp, 2000). The involvement of chromosome 13 in blood pressure salt sensitivity and renal disease was convincingly demonstrated by chromosomal substitution. These observations illustrate the influence of genetic heterogeneity in the traditional linkage studies that tend to mask the effects of specific genes. Similar problems of heterogeneity in the genetic background have also been encountered using gene knockout or inactivation in mice (Dietrich et al. 1993; Threadgill et al. 1995). The potential power of consomic panels for genetic discovery and for the identification of susceptible and resistant strains for mechanistic studies is considerable.

These results from our own laboratory confirm the conclusions regarding the mapping power of consomic approaches reached by Matin et al. (1999), who in search of genes for testicular cancer found that a genome scan provided only modest linkage on several loci that predisposed the mice to testicular germ-cell tumours (TGCTs). Convincing evidence for an important locus on chromosome 19 was obtained only when chromosome 19 was replaced with that from the strain with the wild-type allele. In these mice, the tumour susceptibility of the male mice was reduced from 80% to 5.6% with the chromosomal substitution.

Use of consomic strains to narrow the QTL region of interest within a chromosome by linkage analysis

Once a chromosome of interest has been identified within a consomic panel, the consomic strain can be backcrossed to the parental strain to carry out a linkage analysis in which the QTL related to the trait of interest can be identified within the target chromosome. For example, if BN chromosome 1 was substituted into the SS strain to produce an SS-1BN consomic strain; this consomic strain is then backcrossed to the parental SS strain to produce the F1 generation that would be heterozygous on chromosome 1. These F1 rats would then be brother–sister mated to produce a large F2 population and the trait of interest mapped within chromosome 1. Remember that all chromosomes except chromosome 1 would be fixed in the homozygous state within this backcross and intercrosses, so the recombinations required for mapping would occur only on chromosome 1. The statistical power of linkage analysis becomes much greater in this situation since one does not have to contend with all of the heterozygosity within the entire genome that normally reduces the sensitivity of a linkage study and requires very large population sizes.

Use of consomic strains to narrow the QTL regions of interest by development of overlapping congenics

The development of the consomic panels now brings full circle the original goal that necessitated their development; that is, the need to more rapidly and affordably develop narrowly focused congenic model systems with the substituted region containing a manageable and identifiable number of candidate genes. At the present time, a large number of suggestive QTL regions of interest have been defined. Given the nature of linkage analysis, each of these QTL regions is very broad (∼30 centiMorgans (cm)) and contain hundreds of genes. The challenge of the next phase of discovery is the task of narrowing these loci sufficiently to identify the genes responsible for the observed functional changes in the pathways of interest. Although newly identified QTL are often viewed as representing regions containing candidate genes, it must be recognized that this association needs to be validated and is only a first step in gene identification. Phenotypic differences in the sequence of a candidate gene that alters the expression and/or function of the protein must be identified and a mechanism by which the gene product alters a phenotype must be established. Creation of overlapping strains is needed to achieve these objectives and the consomic panel of rats is the only practical starting point to reduce the timeline of discovery from many to less than one year. Backcrossing the informative recombinant strain to the parental line to narrow the QTL can, when coupled with cDNA expression profiling, yield a narrow region with a reasonable number of candidate genes for identification. Furthermore, the draft sequence of the Brown Norway (BN) rat has now been completed and a bacterial artificial chromosome (BAC) library in the BN rat is currently being built by NHGRI. Such inbred models when used in parallel with these genomic tools can now be effectively utilized to study in greater depth the functional relevance of a small QTL region on the trait of interest.

As such, the consomic rat can be used to generate congenic inbred strains in any particular region of the rat genome within 6–9 months, rather than the standard 3 years by generating an F2 cross with the inbred parental strain (Fig. 3). Multiple overlapping congenics can be bred quickly and generation of about 1000 F2 animals yields, on average, 10 overlapping congenics for every 100 cM. To obtain a rat that was homozygous within a single 1 cm interval, one can roughly estimate that in a population of 1000 F2 rats, there would be, on average, animals that are congenic at a 1–2 cM resolution. Since the three panels of consomic strains capture nearly 50% of the genetic variance present in the 48 most commonly studied strains of inbred rats, these panels alone enable one to construct defined polygenic models and to deconstruct polygenic traits in the parental strains into single locus models as narrow as required to begin identification of a small number of candidate genes (5–20) for positional cloning.

Consomic strains serve as better control strains for the parental model systems

The Dahl salt-sensitive rat has long been of interest to those studying mechanisms responsible for salt-sensitive forms of hypertension. Traditionally, the salt-resistant Dahl R strain has been used as the control strain in these studies, much like the WKY strain has been used as the control strain for the SHR strain of hypertensive rats. These and other control strains were selected based on the common ancestral origins of the strain to be studied, but underwent selection becoming normotensive rather than hypertensive. With the technology for genome scanning available, it became apparent that these ‘control’ strains were at best only 10% different genetically from the inbred hypertensive strains. It has also become apparent by genotyping these rat strains that the hundreds of phenotypic differences distributed throughout the whole genome that were found to exist between the ‘control’ and hypertensive strains were most likely unrelated to the aetiology of the disease. Using nearly 1541 microsatellite markers, we confirmed that there is a 77% allelic difference between BN/Mcwi and SS/Mcwi rats, 48% between SHR and WKY, 52% difference between Sprague Dawley and SS/Mcwi, 57% difference between ACI and SS/Mcwi, and a 30% genetic difference between Dahl R and SS/Mcwi rats (http://rgd.mcw.edu/).

With the emergence of consomic rat strains, it is now possible to identify more relevant control strains that are less genetically different from the parental inbred strains. Studies in our own laboratory, for example, currently use the consomic strain in which chromosome 13 of the BN/Mcwi rat has been substituted into the isogenic background of the parental SS/Mcwi rat (SS-13BN) as the control strain for studies of the SS/Mcwi strain (Cowley et al. 2001; Liang et al. 2002). This consomic strain exhibits significantly lower levels of arterial pressure, proteinuria and renal disease when fed a high salt diet (Cowley et al. 2001). However, the SS-13BN consomic strain exhibits only 1.95% allelic variation from the parental SS/Mcwi strain. Thus, it provides the best available normotensive control strain for physiological comparisons with the hypertensive SS strains.

The rat genome database

The database housing all of the available sequence, mapping and phenotypic data for rats can be accessed at http://rgd.mcw.edu. This database consolidates and integrates data generated from ongoing rat genetic and genomic research efforts making them widely available to the scientific community. This site includes curated data on rat genes, quantitative trait loci (QTL), microsatellite markers from rat strains used in genetic and genomic research. It also contains a dynamic homology tool (VC map) that allows researchers to view mapped genes, their sequences and their locations in rat, mouse, and human organisms. Results obtained from the mouse, rat, and human genome projects can now be largely integrated together by utilizing the powerful bioinformatic tools of comparative genomics, making many physiological studies relevant to the biomedical research community at large (Stoll et al. 2000). By using these tools to develop new animal models and linking genomic information to these models, there are now considerable resources enabling one to attach biological pathways to the genomic infrastructure.

Summary

The development and initial physiological screening of two inbred consomic rat panels (44 strains) on two genetic backgrounds is scheduled to be completed over the next 18 months. These consomic inbred strains provide important advantages in elucidating the relationships of genes to complex biological pathways and disease. They overcome the problems encountered with segregating crosses whereby, even if linkage is found, each individual in the cross is genetically unique and the combination of genes cannot be reproduced or studied in detail. Genomic substitution approaches (either consomic or congenic) provide greater statistical power to detect linkage than traditional F2 crosses. They also provide sufficient numbers of rats to validate the relationship between the trait that segregated and the markers within the QTL region. Finally, these rats can be studied in a replicative manner to elucidate in greater detail the sequential changes responsible for the observed phenotypes of these adult animals. Thus consomic rat strains are proving to be a unique scientific resource that greatly extends our understanding of genes and complex normal and pathological function.

Acknowledgments

The authors would like to thank Meredith M. Skelton, Dr. Carol Moreno and Mary L. Kaldunski for their careful reading of this manuscript. This work was supported by National Heart, Lung, and Blood Institute Grants HL-66579, HL-54998, and HL-29587.

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