Altered Response to Total Body Irradiation of C57BL/6-Tg (CAG-EGFP) Mice

Abstract

Application of green fluorescent protein (GFP) in a variety of biosystems as a unique bioindicator or biomarker has revolutionized biological research and made groundbreaking achievements, while increasing evidence has shown alterations in biological properties and physiological functions of the cells and animals overexpressing transgenic GFP. In this work, response to total body irradiation (TBI) was comparatively studied in GFP transgenic C57BL/6-Tg (CAG-EGFP) mice and C57BL/6 N wild type mice. It was demonstrated that GFP transgenic mice were more sensitive to radiation-induced bone marrow death, and no adaptive response could be induced. In the nucleated bone marrow cells of GFP transgenic mice exposed to a middle dose, there was a significant increase in both the percentage of cells expressing pro-apoptotic gene Bax and apoptotic cell death. While in wild type cells, lower expression of pro-apoptotic gene Bax and higher expression of anti-apoptotic gene Bcl-2, and significant lower induction of apoptosis were observed compared to GFP transgenic cells. Results suggest that presence of GFP could alter response to TBI at whole body, cellular and molecular levels in mice. These findings indicate that there could be a major influence on the interpretation of the results obtained in GFP transgenic mice.

Keywords: GFP transgenic mice; adaptive response; apoptosis; bone marrow death; green fluorescent protein; ionizing radiation.

Figure 1.

Thirty-day survival test after a challenge dose of 7.5 Gy in C57BL/6N-WT mice. Effect of a priming dose of 0.5 Gy X-rays on a subsequent challenge dose of 7.5 Gy X-rays on mouse 30-day survival was verified in both female (A) and male (B) animals. The solid line denotes the animals (4 females and 4 males) that were untreated with TBI. The dotted line represents the animals (30 females and 30 males) that were irradiated with only the challenge dose at postnatal 8 weeks. The broken line stands for the animals (30 females and 30 males) that were primed with a dose of 0.5 Gy X-rays at postnatal 6 weeks followed by a challenge dose of 7.5 Gy X-rays at postnatal 8 weeks. Two asterisks (**) indicate statistically significant differences (P < 0.01) between the 2 groups that were compared.

Figure 2.

Thirty-day survival test after a challenge dose of 7.5 Gy in C57BL/6N-GFP mice. Effect of a priming dose of 0.5 Gy X-rays on a subsequent challenge dose of 7.5 Gy X-rays on mouse 30-day survival was verified in both female (A) and male (B) animals. The solid line denotes the animals (4 females and 4 males) that were untreated with IR. The dotted line represents the animals (25 females and 33 males) that were irradiated with only the challenge dose at postnatal 8 weeks. The broken line stands for the animals (33 females and 33 males) that were primed with a dose of 0.5 Gy X-rays at postnatal 6 weeks followed by the challenge dose at postnatal 8 weeks. Two asterisks (**) indicate statistically significant differences (P < 0.01) between the 2 groups that were compared.

Figure 3.

Number of apoptotic cells in the nucleated bone marrow cells in C57BL/6N-WT and C57BL/6N-GFP mice 6 h after 4.5Gy-TBI. Validation of apoptotic cell appearance was performed by flow cytometry. At least 6 samples from both female and male mice were measured at each time point in each group. The representative histogram images were shown in panel A. Results were shown in bar graphs in panel B. Two asterisks (**) indicate statistically significant differences (P < 0.01) between the 2 groups that were compared.

Figure 4.

Change of cell cycle phases in the nucleated bone marrow cells in C57BL/6N-WT and C57BL/6N-GFP mice 6 h after 4.5Gy-TBI. Validation of cell cycle phases in the cells was performed by flow cytometry. At least 6 samples from both female and male mice were measured at each time point in each group. The representative histogram images were shown in panel A. Results were shown in bar graphs in panel B. One asterisk (*) and 2 asterisks (**) indicate respectively statistical differences at P < 0.05 and P < 0.01 between the 2 groups that were compared.

Figure 5.

Percentage of positive cells expressing tested proteins in the nucleated bone marrow cells in C57BL/6N-WT (A) and C57BL/6N-GFP (B) mice 3 h after 4.5Gy-TBI. Validation of positive immunohistochemically stained cell appearance in the cells was performed flow cytometrically. At least 6 samples from both female and male mice were measured in each group. One asterisk (*) indicates statistical differences at P < 0.05 between the 2 groups that were compared.