The development and biological characteristics of a novel potentially radioresistant inbred mouse strain

Abstract

The growth of biomedical research over the previous decades has been accompanied by an increase in the number, complexity and diversity of experimental animals developed as research tools, and inbred mice are some of the most widely used. However, thus far, no inbred mice have exhibited strong radioresistance for use in radiation‑damage research. To develop a radioresistant mouse model, a female Japanese outbreeding strain ICR/JCL mouse was mated with a male Chinese inbred strain 615 mouse. From the F1 generation, the mouse line was maintained by brother‑to‑sister mating. A novel mouse strain was established over >20 continuous generations and designated the Institute of Radiation Medicine-2 (IRM-2) mouse. The biological characteristics, genetic characteristics and susceptibility to radiation of these mice were determined. The IRM‑2 mice inherited traits from the parents, including strong reproductive capacity, stable physiological and biochemical indices and few differences among individuals. According to the genetic results, the IRM‑2 mice exhibited homozygosity, isogenicity and consistency, in agreement with international standards for inbred strains. Radiosensitivity studies have previously suggested that the lethal dose (LD)50 values for IRM‑2 mice were 7.17 Gy (male) and 7.5 Gy (female), resulting in a dose reduction factor value of 1.39 (male) and 1.37 (female). The mortality of IRM‑2 mice irradiated with 8 Gy total body irradiation was 15% at day 9 and 90% at day 15 after radiation. The number of nucleated cells in bone marrow, DNA content and colony‑forming unit‑spleen counts in IRM‑2 mice after exposure to γ‑ray irradiation were markedly higher than the corresponding values for the parental strains, suggesting that the IRM‑2 mice exhibit high resistance to ionizing radiation. Thus, it is suggested that this novel inbred mouse strain may be developed as an animal model of radioresistance for future use in radiation research.

Figure 1.

An IRM-2 mouse and its parents. A novel mouse strain was derived from crosses between ICR/JCL mouse (maternal line) and 615 mouse (parental line) and designated the IRM-2 mouse through more than 20 continuous breeding generations. (A) IRM-2 mouse and (B) ICR/JCL mouse (maternal strain) and 615 mouse (parental strain).

Figure 2.

Growth curves of IRM-2 mice. The growth and development of IRM-2 mice was determined from birth and the growth curves were plotted (n=40). The increases in (A) body weight, (B) body length and (C) tail length were measured from birth to 70 days.

Figure 3.

Peripheral blood and bone-marrow characteristics of IRM-2 mice. Whole blood was drawn from the orbital sinuses of 7-week-old and 14-week-old male and female IRM-2 mice to count peripheral blood cells. BMCs were flushed from 7-week-old and 14-week-old male and female IRM-2 mice femurs and counted (n=10). (A) Peripheral WBC count of IRM-2 mice. (B) Peripheral RBC count of IRM-2 mice. (C) Peripheral PLT count of IRM-2 mice. (D) BMC count of IRM-2 mice. BMCs, bone-marrow cells; WBC, white blood cell; RBC, red blood cell; PLT, platelet.

Figure 4.

Coat color of F1-generation mice hybridized from IRM-2 and BALB/c parents. An IRM-2 mouse was mated with a BALB/c mouse with known genes. Coat genotype of IRM-2 mice was identified based on the coat color of two-week-old F1-generation mice.

Figure 5.

Chromosome karyotype and relative chromosome length in the IRM-2 mice. IRM-2 mice were administered an intraperitoneal dose of 7 µg/g body weight colchicine and euthanized after 4 h. (A) Bone-marrow cells were flushed from the femurs. Metaphase slides were prepared by dropping the cells onto glass slides, digesting with 0.03% trypsin-EDTA solution and staining with Giemsa. (A) Typical G-banding metaphase was observed (magnification, ×400). (B) G-banding karyotype was drawn based on G-banding patterns of chromosomes (original magnification, ×400). (C) Chromosomes were amplified according to G-banding karyotype. The lengths of the straighter chromosome from each pair homologous chromosomes were measured and relative chromosome lengths were calculated.

Figure 6.

LD₅₀ of various mice strains irradiated with ¹³⁷Cs γ-radiation. BALB/c, C57BL/6, ICR/JCL, 615 and IRM-2 mice were exposed to total body irradiation of 0–10 Gy (absolute lethal dose) ¹³⁷Cs γ-rays and the mortality of each group was recorded 30 days after irradiation to calculate the LD₅₀ for the mice (n=10). **P<0.01 vs. IRM-2 mice. LD, lethal dose.

Figure 7.

Survival curve of mice after total body irradiation with 8 Gy doses of ¹³⁷Cs γ-rays. The number of surviving mice was recorded continuously for 15 days after exposure to radiation and the survival curve of mice was determined (n=20). The data were expressed as percentage of surviving mice.

Figure 8.

Changes in the number of leukocytes and in the hematopoietic function of mice irradiated with γ-radiation. The function of the hematopoietic system was examined to test the radiosensitivity of IRM-2 mice. IRM-2, ICR/JCL and 615 mice were irradiated with 4 Gy γ-radiation (n=8). (A) Whole blood was extracted from the orbital sinuses on 2, 5, 9, 15 and 21 days after irradiation and the leukocytes were counted. *P<0.05 vs. IRM-2 mice 12 to 21 days after radiation. (B) Whole blood was extracted from the orbital sinuses on 2, 5, 9, 15 and 21 days after irradiation and the BMNCs were counted. (C) BMCs were flushed from mouse femurs on 2, 5, 9, 15 and 21 days after irradiation and treated with a 0.2 mol/l HClO₄ solution. The DNA content of the BMCs was measured at 286 nm. (D) Spleens were harvested from the abdominal cavity of mice on 2, 5, 9, 15 and 21 days after irradiation and CFU-S was counted under a microscope. *P<0.05, **P<0.01 vs. IRM-2 mice 9 to 21 days after radiation. BMNCs, bone marrow nucleated cells; BMCs, bone marrow cells; CFU-S, colony-forming unit-spleen.