Abstract
Purpose
To confirm the presence of the susceptible gene for the thymoma development in the region that was assumed by the previous linkage study by Oyabu et al. (J Natl Cancer Inst 91:279–282, 1999), we tried to establish a congenic strain of rats.
Methods
Backcrossings between the BUF/Mna strain as a donor strain and the ACI/NMna strain as an inbred partner were repeated for 12 generations, examining whether rats had the thymoma development region, and then homozygous rats were yielded by mating among the heterozygotes. To detect the phenotypic expression, heterozygous ACI.BUF/Mna-Tsr1/+ (ACI-Tsr1/+) rats were generated by crossing female ACI.BUF/Mna-Tsr1/Tsr1 (ACI-Tsr1/Tsr1) rats with male ACI/NMna rats and were maintained for 24 months.
Results
These ACI-Tsr1/+ rats produced thymoma in 71%, showing a dominant trait. The thymomas were of the lymphocyte predominant type, as those developed in rats of the original BUF/Mna strain.
Conclusions
Thus, a new rat congenic strain, ACI-Tsr1/Tsr1, was established, revealing that thymoma develops in the dominant trait in ACI-Tsr1/+ rats.
Keywords: Rat model, Thymoma, ACI.BUF/Mna-Trs1/Tsr1
Introduction
The BUF/Mna strain is a susceptible line of rats for the spontaneous development of the thymoma (Matsuyama et al. 1972; Yamada et al. 1973; Matsuyama et al. 1975, 1988 Matsuyama and Nagayo 1977; Matsuyama 1990; Taguchi et al. 1992). Linkage study revealed that thymoma susceptibility was determined by an autosomal dominant locus, thymoma susceptible locus of rat-1 (Tsr1) (Matsuyama et al. 1986). Genetic mapping of Tsr1 showed that it is located at 4.8 centiMorgans distal to the molecular marker D7Rat21 on chromosome (Chr.) 7 (Oyabu et al. 1999). The Tsr1 region narrowed down into the region between D7Wox16 and D7Rat21 (Higo et al. 2000). Therefore, we attempted to establish a congenic thymoma-prone strain of rat by placement of the Tsr1 region into the genome of the ACI/NMna strain.
Materials and methods
Establishment of the ACI.BUF/Mna-Tsr1/Tsr1 (ACI-Tsr1/Tsr1) congenic strain
Crosses were begun between the BUF/Mna strain as a donor and the ACI/NMna strain as an inbred partner at the Institute of Animals Diseases, Fujita Health University in 1997. Heterozygous female rats (ACI-Tsr1/+) were identified by molecular tests of ear biopsies before being used in the backcrossing. After 12 generations of backcrossing to the ACI/NMna strain, a new congenic strain, ACI-Tsr1/Tsr1, was established by mating among heterozygous littermates. In each generation, rats were confirmed the possession of the Tsr1 region, which was flanked by D7Wox16 and D7Rat69 loci. The congenic strain is maintained by a standard brother-sister mating protocol to render a segment bearing the Tsr1 locus homozygous. The use of these rats in the experimental protocols described was approved by the Fujita Health University Animal Care Committee.
Genotyping of Tsr1 locus
Genomic DNAs were prepared from ear biopsies of the rats using the automatic DNA purification system (PI-200, Kurabo, Japan). Genotyping of Tsr1 locus was carried out by standard polymerase chain reaction (PCR) with five single sequence length polymorphism (SSLP) markers, D7Wox16, D7Rat53, D7Rat21, D7Rat142 and D7Rat69 (Serikawa et al. 1992, Oyabu et al. 1999).
Detection of phenotype of the Tsr1 locus
Heterozygous ACI-Tsr1/+ rats were raised by crossing female ACI-Tsr1/Tsr1 rats with male ACI/NMna rats, and maintained for 24 months to detect the development of thymoma. The heterozygous ACI-Tsr1/+ rats were used to confirm the dominant trait of the Tsr1 locus to the development of thymoma. All of the rats were killed at the age of 24 months, and complete autopsies were performed. Tumors were fixed by 10% formalin, embedded in paraffin, cut, and stained with hematoxyline and eosin.
Results
Six out of eight female (75%) and four out of six male (67%) ACI-Tsr1/+ rats developed thymoma as nodules in the involuted thymuses (Fig. 1; Table 1). No sex difference was found in the incidence. All of the thymomas were of lymphocyte predominant type, corresponding to type B1 of human thymoma (Palestro et al. 2004). A few epithelial cells with large nuclei and eosinophilic cytoplasm were interspersed in the accumulation of lymphocytes (Fig. 2). Very rarely mitotic epithelial cells were discernible. On the other hand, no thymoma was found in rats of the background ACI/NMna strain. A few other types of tumors were found (Table 1), as previously reported in the (BUF/Mna × ACI/NMna)F1 rats (Matsuyama et al. 1988).
Fig. 1.
A round thymoma nodule developed in an involuted thymus lobe of an ACI/NMs-Tsr1/+ rat. Parathymic lymph nodes are also seen (upper and lower rights). Image at 4× magnification
Table 1.
Incidence of thymoma and other tumors in ACI-Tsr1/+ and ACI/NMna rats
| Strain | Sex | No. of rats survived for 104 weeks | No. of rats with thymoma | No. of rats with other tumors* |
|---|---|---|---|---|
| ACI-Tsr1/+ | Female | 8 | 6 | 2 |
| ACI-Tsr1/+ | Male | 6 | 4 | 4 |
| ACI | Female | 9 | 0 | 4 |
| ACI | Male | 7 | 0 | 3 |
*Contained rats with tumors of the pituitary, adrenal, and mammary glands
Fig. 2.
High power microscopic feature of the thymoma shown in Fig. 1. A few large epithelial cells are seen in an accumulation of lymphocytes. Image is at 40× magnification
Discussion
We report here the establishment of a new congenic rat strain, ACI-Tsr1/Tsr1, and describe the characteristics of the spontaneously developed thymoma. The length of the donor chromosome of Chr. 7 is genetically defined. Dominant trait of the Tsr1 region was also confirmed in this study. Thus, this congenic strain of rat will be useful for elucidation of the mechanism of the development of thymoma, analyzing the abnormality of genes which locate within the region of the Tsr1, which was defined by the previous linkage studies (Oyabu et al. 1999; Higo et al. 2000). It is noteworthy that many genetic aberrations, including losses of heterozygosity and amplifications, were found in loci of Chr. 8 in human thymomas (Inoue et al. 2003), since many of loci of human Chr. 8 are synthenic to those of rat Chr. 7 (Yamada et al., 1994; Serikawa et al., 1998).
Rats of the BUF/Mna strain developed hyperplasia of the thymus in the early life, as an expression of thymus enlargement loci (Ten1 and Ten2) (Murakumo et al. 1996). However, the thymomas were developed as a nodule in the involuted thymuses in the ACI-Tsr1/+ rats. Rats of the background ACI/NMna strain have no Ten-1 nor Ten-2 regions, it is therefore, reasonable that thymomas developed as a discrete nodules in involuted thymuses.
The thymoma developed in the ACI-Tsr1/Tsr1 strain had quite similar microscopic features to those of the type B1 of human thymoma (Palestro et al. 2004), so it is good animal model for human thymoma, having a strict control ACI/NMna strain.
Acknowledgments
We thank Prof. T. Serikawa for his advice, and Miss K. Ishikawa and Miss J. Adachi for their technical assistance. This work was supported by the Imanaga Medical Foundation. We are also thankful to the National Bioresource-Rat for keeping rats of the congenic strain.
Abbreviations
- Tsr1
Thymoma susceptible locus of rat-1
- ACI-Tsr1/Tsr1
ACI.BUF/Mna-Tsr1/Tsr1
- ACI-Tsr1/+
ACI.BUF/Mna-Tsr1/+
- Chr
Chromosome
- PCR
Polymerase chain reaction
- SSLP
Single strand length polymorphism
- Ten-1
Thymus enlargement locus-1
- Ten-2
Thymus enlargement locus-2
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