Congenic Strains for Genes Affecting
Our work involves a mouse model for susceptibility to carcinogen-induced colon cancer in mice. Strain ICR/Ha is highly sensitive to colon tumors using the carcinogen 1,2-dimethylhydrazine dihydrochloride (DMH) or its metabolite azoxymethane (AOM), while C57BL/6Ha is resistant. QTL analysis of a backcross involving these strains identified loci on Chrs 5, 12, 13, and 14 containing genes affecting the number of tumors produced after treatment. The goal is to identify the genes involved.
Congenic strains have been made for all four regions, each containing a long congenic interval from the susceptible strain ICR on a C57BL/6Ha background. After DMH treatment, each of the strains developed tumors. Multiple subcongenic strains, with shorter intervals from ICR, have been constructed for two of the genetic regions, and have been made homozygous. These strains are undergoing a carcinogen challenge. Subcongenic construction is almost complete for the other strains. Mlh3 is a candidate for the susceptibility gene on Chr 12, but no difference in the cDNA sequence between the two strains has been identified. The subcongenic strains will help determine if the genetic region containing the susceptibility gene also contains Mlh3.
Loci Affecting Hybrid Sterility in Male Mice
Project 1. M. spretus
Interspecific hybrid males from C57BL/6 andM. spretus parents are infertile. Verne Chapman, Yoichi Matsuda and others have shown that this infertility is associated with a lack of X-Y pairing in the pseudoautosomal region. However, there is evidence that other genes on proximal Chr X may be involved. A genetic resource (AT24), developed by Dr. Chapman, useful for the analysis of hybrid sterility, is a congenic strain, C57BL/6.SPRET-Hprts. In this strain, a portion of Chr X fromM. spretus was transferred to a C57BL/6 background. The region fromM. spretus is 17 map units long, and extends fromDXMit48 toDXMit60. Males from this congenic strain have small testes (0.11 g vs. 0.21 g) in which only half of the tubules contain developing sperm. The males are semifertile, but do breed. The locus responsible for this phenotype has been namedIhtw1 (interspecific hybrid testis weight 1).
Progress: Project 1.
Using subcongenic strains to subdivide the congenic interval fromM. spretus, the locusIhtw1 has been localized to a 1 cM region within the original congenic strain. We have constructed a new congenic strain with just this short region fromM. spretus. Testis weight in this strain is 0.15 g, suggesting that there is a second locus within the congenic region that affects testis weight. The mouse sequence in this region is being screened for candidate.
A screen for suppressors of the mutation has identified 2 genetic regions likely to contain a suppressor. One is on proximal Chr 19, the other on central Chr 11. Experiments to more closely define these regions are in progress.
The congenic strain, AT24, is also associated with large placentas (0.2 g, vs. 0.1 g). These large placentas have an elevated level of spongiotrophoblast cells and glycogen. There is a good correlation between placental weight and the length of the region from M. spretus in the congenic line. Furthermore, an influence of fetal sex on the placental development has been observed, as placentas attached to males tend to be larger than those attached to females. The data suggest that the sex difference of placental weights in these hybrids is caused by interactions with the Y chromosome. This study has been done in collaboration with Dr. Fundele of the Max Planck Institute in Berlin.
Project 2. M. macedonicus
Interspecies hybrids involvingM. macedonicus have testes weighing only 0.05 gm. They do not produce sperm, and cytological studies show that meiosis rarely reaches the pachytene stage in adults. Histology shows that testis tubules contain Sertoli cells and mitotically dividing spermatogonia. The rest of the tubule contains vacuoles. Our genetic analysis of the phenotype, using QTL analysis of a backcross and subsequent generation of a congenic strain, suggests that one or more loci on proximal Chr X and on proximal Chr 17 need to be hybrid to generate this phenotype. Our goal is to identify the genes involved.
Progress: Project 2.
Chr X: Histology of juvenile testes for hybrids, where the meiotic process is synchronous, shows that in the hybrid congenic strain, progression through meiosis begins normally at day 8. By day 15, pachytene paired chromosomes can be seen, but the meiotic cells are contained in vacuoles. At about day 21 many of the meiotic cells are destroyed, with residual contents in large vacuoles. This process continues to adulthood. Subcongenic analysis shows that a number of genetic regions contribute to the weight decrease, as well as to the numbers of tubules containing vacuoles. This is clearly a complex phenotype, and careful phenotypic analysis will be required to identify the genes involved. Analysis for apoptosis using the TUNEL method has indicated that apoptosis is occurring as the spermatocytes are destroyed, both in juvenile and adult testes.
Chr 17: Congenic hybrids involving Chr 17 progress through meiosis and spermatogenesis, but no sperm tails are visible and the testis tubules become plugged with sperm heads. Testis weight is about 0.15 gm. Subcongenic strains have been analyzed for testis weight and histology. These data have indicated a critical region of 2.5 Mb containing the gene causing the phenotype. A strong candidate is the dynein gene that has also been implicated in hybrid sterility 6.
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