Identification of differentially regulated genes in mildly hyperlipidemic ApoE3-Leiden mice by use of serial analysis of gene expression.

Although genes determining lipoprotein homeostasis and atherosclerosis are the subject of intensive investigation, only a subset of these genes is known at present. Hence, we do not have sufficient knowledge to explain the genetic basis of hyperlipidemia in the majority of subjects. Our aim was to identify novel genes and pathways underlying lipoprotein homeostasis by using serial analysis of gene expression. The liver expression profile of mild hyperlipidemic apolipoprotein E3-Leiden (E3L) transgenic mice was compared with that of the wild-type C57BL/6JIco (B6) mice. Over 18 000 liver transcripts of B6 as well as E3L mice were analyzed, representing >9400 unique genes. One hundred seventy-five genes showed altered expression between the strains (P<0.05). Although several of these genes belonged to known metabolic pathways, such as lipoprotein metabolism, detoxification processes, glycolysis, and the acute-phase response, most were novel. Differential gene expression of 8 of 10 genes tested could be confirmed by Northern blot analysis. This inventory of differentially expressed genes will provide a unique basis for detailed studies to gain more insight into their role in lipoprotein homeostasis and atherosclerosis.

Four new colon cancer susceptibility loci, Scc6 to Scc9 in the mouse.

Germ-line mutations in APC and mismatch repair genes explain only a small percentage of all colorectal cancer cases. We have used the recombinant congenic strain mouse model to find new loci that are involved in the control of susceptibility to colon cancer. Five different colon cancer susceptibility genes, Scc1-Scc5, have been described previously using the recombinant congenic strains. Two of these loci, Scc4 and Scc5, show a reciprocal, genetic interaction. Here we report the mapping of four new colon tumor susceptibility genes: (a) Scc6 on chromosome 11; (b) Scc7 on chromosome 3; (c) Scc8 on chromosome 8; and (d) Scc9 on chromosome 10. Scc7 and Scc8 show a genetic interaction; Scc7 is only detected by virtue of its interaction with Scc8.

Simulation of the distribution of parental strains' genomes in RC strains of mice.

Recombinant Congenic strains (RC strains) were developed to facilitate mapping of genes influencing complex traits controlled by multiple genes. They were produced by inbreeding of the progeny derived from a second backcross from a common 'donor' inbred strain to a common 'background' inbred strain. Each RC strain contains a random subset of approximately 12.5% of genes from the donor strain and 87.5% of genes from the background strain. In this way the genetic control of a complex disease may be dissected into its individual components. We simulated the production of the RC strains to study to what extent they have to be characterized in order to obtain sufficient information about the distribution of the parental strains' genomes in these strains and to acquire insight into parameters influencing their effectiveness in mapping quantitative trait loci (QTLs). The donor strain genome in the RC strains is fragmented into many segments. Genetic characterization of these strains with one polymorphic marker per 3.3 centiMorgans (cM) is needed to detect 95% of the donor strain genome. The probability of a donor strain segment being located entirely in between two markers of background strain origin that are 3 cM apart (and hence escaping detection) is 0.003. Although the donor strain genome in the RC strains is split into many segments, the largest part still occurs in relatively long stretches that are mostly concentrated in fewer than 13 autosomes, the median being 9 autosomes. Thus, in mapping QTLs, the use of RC strains facilitates the detection of linkage.

Gene interaction and single gene effects in colon tumour susceptibility in mice.

To dissect the multigenic control of colon tumour susceptibility in the mouse we used the set of 20 CcS/Dem (CcS) recombinant congenic (RC) strains. Each CcS strain carries a unique, random subset of approximately 12.5% of the genome of strain STS/A (STS) on the genetic background of BALB/cHeA (BALB/c). Previously, applying a protocol of 26 injections of 1,2-dimethylhydrazine (DMH), we detected two susceptibility loci, Scc1 and Scc2, on chromosome 2 (refs 4, 5). Using a shorter tumour-induction procedure, combining DMH and N-ethyl-N-nitrosourea (ENU) treatment, we demonstrate that BALB/c, STS and most CcS strains are relatively resistant. The strain CcS-19, however, is susceptible, probably due to a combination of BALB/c and STS alleles at several loci. Analysis of 192 (BALB/c x CcS-19) F2 mice revealed, in addition to the Scc1/Scc2 region, three new susceptibility loci: Scc3 on chromosome 1, Scc4 on chromosome 17 and Scc5 on chromosome 18. Scc4 and Scc5 have no apparent individual effect, but show a strong reciprocal interaction. Their BALB/c and STS alleles are not a priori susceptible or resistant but the genotype at one locus determines the effect of the allele at the second locus and vice versa. These findings and the accompanying paper on lung tumour susceptibility show that interlocus interactions are likely to be an important component of tumour susceptibility.

Different genetic susceptibility to aberrant crypts and colon adenomas in mice.

Aberrant crypt foci (ACFs) are the earliest identifiable epithelial lesions thought to precede the development of a subset of colon tumors. To assess their predictive value to adenoma development, we have tested in mice whether the development of ACFs and adenomas is controlled by the same genes. Therefore we used the CcS/Dem series of recombinant congenic strains, in which the effect of multiple susceptibility genes might be studied separately. We investigated susceptibility to ACFs in nine CcS/Dem strains and their parental strains, BALB/cHeA and STS/A, 4 weeks after s.c. injection of 1,2-dimethylhydrazine (20 mg/kg body weight). For the strains BALB/cHeA, STS/A, and CcS-19, we also examined the number of ACFs 2, 8, and 12 weeks after treatment. Susceptibility to adenomas was measured as the number of adenomas 6 weeks after 26 weekly s.c. injections of 1,2-dimethylhydrazine (15 mg/kg body weight). The nine CcS/Dem strains, the BALB/ cHeA strain, and the STS/A strain exhibited different patterns of susceptibility to ACFs and adenomas, demonstrating that different subsets of susceptibility genes are involved. Therefore, in evaluating the role of ACFs as a predictive marker for adenoma development, genetic factors must be taken into account.

Fine mapping of colon tumor susceptibility (Scc) genes in the mouse, different from the genes known to be somatically mutated in colon cancer.

The predisposition to colon cancer is multigenetically controlled in animals and probably also in humans. We have analyzed the multigenic control of susceptibility to 1,2-dimethylhydrazine-induced colon tumors in mice by using a set of 20 homozygous CcS/Dem recombinant congenic strains, each of which contains a different random subset of approximately 12.5% of genes from the susceptible strain STS/A and 87.5% of genes from the relatively resistant strain BALB/cHeA. Some CcS/Dem strains received the alleles from the susceptible strain STS/A at one or more of the multiple colon tumor susceptibility loci and are susceptible, whereas others are resistant. Linkage analysis shows that these susceptibility genes are different from the mouse homologs of the genes known to be somatically mutated in human colon cancer (KRAS2, TP53, DCC, MCC, APC, MSH2, and probably also MLH1). Different subsets of genes control tumor numbers and size. Two colon cancer susceptibility genes, Scc1 and Scc2, map to mouse chromosome 2. The Scc1 locus has been mapped to a narrow region of 2.4 centimorgans (90% confidence interval).

The recombinant congenic strains for analysis of multigenic traits: genetic composition.

The genetic control of susceptibility to many common diseases, including cancer, is multigenic both in humans and in animals. This genetic complexity has presented a major obstacle in mapping the relevant genes. As a consequence, most geneticists and molecular biologists presently focus on "single gene" diseases. To make the multigenic diseases accessible to genetic and molecular analysis, we developed a novel genetic tool, the recombinant congenic strains (RCS) in the mouse (4). The RC strains are produced by inbreeding of mice of the second backcross generation between two inbred strains, one of which serves as the "donor" and the other as the "background" strain. A series of RCS consists of approximately 20 strains, each carrying a different set of genes: approximately 12.5% genes from the common donor inbred strain, the remaining 87.5% from the common background inbred strain. As the set of donor strain genes in each RC strain is different, the nonlinked genes of the donor strain involved in the control of a multigenic trait, e.g., cancer susceptibility, become distributed into different RC strains where they can be analyzed one by one. Hence, the RCS system transforms a multigenic trait into a series of single gene traits, where each gene contributing to the multigenic control can be mapped and studied separately. Recently we demonstrated that the RCS system is indeed capable of resolving multigenic traits, which are hardly analyzable otherwise, by mapping four new colon tumor susceptibility loci (8; P. C. Groot, C. J. A. Moen, W. Dietrich, L. F. M. van Zutphen, E. S. Lander, and P. Demant, unpublished results). For successful application of the RCS system, extensive genetic characterization of the individual recombinant congenic strains is essential. In this paper we present detailed information about the genetic composition of three series of RC strains on the basis of typing of 120-180 markers distributed along all autosomes. The data indicate that the relative representation of the donor strain genes in the RC strains does not deviate from the theoretical expectation, and that the RC strains achieved a very high degree of genetic homogeneity and for all practical purposes can be considered inbred strains. The density and distribution of markers reported here permits an effective mapping of unknown genes of donor strain origin at almost all autosomal locations. Much of this information has been obtained using the new class of genetic markers, the simple sequence repeat polymorphisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Scc-1, a novel colon cancer susceptibility gene in the mouse: linkage to CD44 (Ly-24, Pgp-1) on chromosome 2.

Mutations of proto-oncogenes and tumor-suppressor genes lead to neoplastic development. Some germline mutations of these genes increase the tumor susceptibility of their carriers, but the relationship between genes controlling tumor susceptibility and the known oncogenes and tumor-suppressor genes remains unelucidated. Moreover, as tumor susceptibility in mouse is controlled by multiple genes, their identification has been virtually impossible. We therefore developed a new system, the recombinant congenic strains (RCS), which separates individual susceptibility genes into different RC strains, thus facilitating their analysis. To map genes controlling the development of colon cancer, we used the Balb/c-c-STS (CcS/Dem) RC strains. Owing to several unidentified genes, Balb/cHeA mice are relatively resistant and STS/A mice highly susceptible to 1,2-dimethylhydrazine-(DMH)-induced colon adenocarcinomas. Each CcS/Dem strain carries a different subset of about 12.5% of genes of the STS strain on the Balb/c background, and individual STS susceptibility genes became segregated into different RC strains. Using CcS-19, one of the highly susceptible RC strains, we mapped a novel colon tumor susceptibility gene, Scc-1, different from the oncogenes and tumor-suppressor genes known to be involved in colon tumorigenesis, in the vicinity of CD44 (Ly-24, Pgp-1) on chromosome 2. The mapping of the Scc-1 gene indicates that the RCS system can be used to map and study the presently unknown genes which control cancer development.

The recombinant congenic strains--a novel genetic tool applied to the study of colon tumor development in the mouse.

The development of tumors in mice is under multigenic control, but, in spite of considerable efforts, the identification of the genes involved has so far been unsuccessful, because of the insufficient resolution power of the available genetic tools. Therefore, a novel genetic tool, the RC (Recombinant Congenic) strains system, was designed. In this system, a series of RC strains is produced from two inbred strains, a "background" strain and a "donor" strain. Each RC strain contains a different small subset of genes from the donor strain and the majority of genes from the background strain. As a consequence, the individual genes of the donor strain which are involved in the genetic control of a multigenic trait, become separated into different RC strains, where they can be identified and studied individually. One of the RC strains series which we produced is made from the parental strains BALB/cHeA (background strain) and STS/A (donor strain). We describe the genetic composition of this BALB/cHeA-C-STS/A (CcS/Dem) series and show, using 45 genetic autosomal markers, that it does not deviate from the theoretical expectation. We studied the usefulness of the CcS/Dem RC strains for analysis of the genetics of colon tumor development. The two parental strains, BALB/cHeA and STS/A, are relatively resistant and highly susceptible, respectively, to the induction of colon tumors by 1,2-dimethylhydrazine (DMH). The individual RC strains differ widely in colon tumor development after DMH treatment; some are highly susceptible, while others are very resistant. This indicates that a limited number of genes with a major effect are responsible for the high susceptibility of the STS strain. Consequently, these genes can be mapped by further analysis of the susceptible RC strains. The differences between the RC strains were not limited to the number of tumors, but the RC strains differed also in size of the tumors and the relative susceptibility of the two sexes. Our data indicate that the number of tumors and the size of tumors are not controlled by the same genes. The genetics of these different aspects of colon tumorigenesis can also be studied by the RC strains. The DMH-treated mice of the parental strains and the RC strains also developed anal tumors and haemangiomas in varying numbers. The strain distribution pattern (SDP) of susceptibility for each of the three types of tumors induced by DMH is different, indicating that development of these tumors is under control of different, largely non-overlapping, sets of genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of the MHC on hormonal induction of mammary tumors and function of hypophyseal isografts in the mouse.

While the role of the H-2 complex in the resistance to virally induced tumors has been extensively studied, little is known about its influence on the development of epithelial tumors of non-viral etiology, although such tumors are most prevalent in humans. Therefore, we analyzed the role of the H-2 complex in susceptibility to mammary tumors induced by hormonal stimulation from heterotopic hypophyseal isografts in H-2 congenic strains from C57BL/10, BALB/c, and O20/A backgrounds. This method of induction allows an assessment of the effect of H-2 genes on the function of various organs involved in this process. We found that the tumor susceptibility genes map to two segments: I-E-S, and to the right of S. The mechanisms by which the H-2 complex affects the induction of mammary tumors in C57BL/10 congenic strains seem to include an influence on several factors involved in the hormonal stimulation, because the susceptible B10 congenic strains have higher plasma levels of prolactin and the H-2 complex also affects the growth of hypophyseal isografts. Their size correlates with tumor development in individual mice in the resistant C57BL/10 congenic strains. We reported previously H-2-dependent differences in levels of the estrogen receptor in hypophysis. For this study, we measured the levels of estrogen receptors in uteri to asses the tissue specificity of this effect of H-2. However, no influence of the H-2 complex on estrogen receptor levels was observed in uteri. Strains from BALB/c and O20 backgrounds developed mammary tumors much earlier than the B10 congenic strains, indicating a strong influence of non-H-2 genes.

Effects of freezing and freeze-drying on the biomechanical properties of rat bone.

The effects of various preservation techniques on the biomechanical properties of bone have been investigated in a rat model. Freezing of the specimens to -20 degrees C, -70 degrees C, and -196 degrees C did not adversely affect the strength of long bones tested in torsion or of vertebral bodies tested in compression. Freeze-drying did not markedly affect the compression properties of the vertebral specimens; however, it did produce a significant deleterious reduction in the torsional strength of the long bones.