Application of chromosomal substitution techniques in gene-function discovery

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.

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

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.

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

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.

Figure 3. Generation of congenic rats from consomic strains

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.

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

Substitution of chromosome 13 from the BN rat into SS strains (SS.13^BN) or chromosome 18 (SS.18^BN), 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.