Generation of a novel transgenic mouse model for bioluminescent monitoring of survivin gene activity in vivo at various pathophysiological processes: survivin expression overlaps with stem cell markers

Abstract

Survival has been implicated to play an important role in various pathophysiological processes. However, because of a lack of appropriate animal models, the role and dynamic expression of survivin during pathophysiology are not well defined. We generated a human survivin gene promoter-driven luciferase transgenic mouse model (SPlucTg) so that dynamic survivin gene activity can be monitored during various pathophysiological conditions using in vivo imaging. Our results show that, consistent with survivin positivity in testis, luciferase activity in normal SPlucTg mice was detected in the testis of male mice. Furthermore, similar to the known requirement of transient expression of survivin for pathophysiological responses, we observed a transient luciferase expression in castrated SPlucTg male mice after supplement of androgen. Significantly, it was reported that survivin expression turns on during mouse liver injury and regeneration; a transient and dose-dependent luciferase expression in the mouse liver was observed after administration of carbon tetrachloride into SPlucTg mice. We further demonstrated that luciferase activity closely correlates with endogenous survivin expression. We also demonstrated that only a subset of cells expresses survivin, and its expression overlaps with the expression of several stem cell markers tested. Thus, we have generated a unique animal model for analysis of diverse pathophysiological processes and possible stem cell distribution/activity in vivo.

Figure 1

Generation of the human survivin promoter-driven luciferase reporter-transgenic (SPlucTg) mice. A: Diagram of the structure of the transgene cassette. Four primers used in PCR to determine genotype of the transgenic mouse are shown. B: Representative genotyping results for the transgenic mice. C: The luciferase expression profile in genotype-positive SPlucTg mice was determined by in vivo imaging. Red arrows indicate testes that show luciferase activity. ♂ indicates male mice; ♀, female mice.

Figure 2

Human survivin promoter-driven luciferase activity (green arrow) during male SPlucTg mouse castration and regrowth with androgen replacement. A: Testosterone pellets were imbedded subcutaneously on day 0 (black arrow) onto SPlucTg mice, which were castrated 14 days ago. Luciferase activity was monitored by in vivo Imaging. The red arrow indicates testes showing luciferase activity before castration (day −15). B: The quantitative data derived from A are presented in a histogram format.

Figure 3

Human survivin promoter-driven luciferase activity (green arrow) during CCl4-induced SPlucTg mouse liver injury and repair processes. A–D: A 10% (v/v) of CCl4 solution in corn oil was injected intraperitoneally on Day 0. Administered doses and the respective imaging day are indicated, respectively. The red arrow points the location of the testes, which are known to be positive for luciferase activity. E–H: The quantitative data derived from A–D are presented in histogram formats, respectively.

Figure 4

Association of luciferase activity with endogenous survivin expression in the CCl4-induced liver injury and repair in SPLucTg mouse model (Line 2). A 10% of CCl4 solution in corn oil at a dose of 200 μl/25 g was injected intraperitoneally on Day 0. Mice were sacrificed and livers were isolated on days 1, 2, 3, 4, and 5, respectively. After the isolated livers were lysed, luciferase activity (upper panel, mean of triplicate assays from same sample) and survivin expression (lower panel) were determined using luciferase activity assays and Western blot analysis, respectively. Examples of mice from each day were shown. HeLa cell lysates was used as a survivin positive control, and the molecular size of the protein markers in the protein marker lane was indicated. m indicates male mice; f, female mice.

Figure 5

The pattern of survivin expression during liver injury and repair overlaps with stem cell markers. A and B: The treated condition is 5% CCl4, 400 μl/25g. Normal liver and CCl4-treated liver tissues were isolated 65 hours after treatment and analyzed by IHC as shown. An example vessel is indicated by the arrow. The liver tissues in A were fixed with buffered formalin, which works well for survivin and CD133 antibodies. The liver tissues in (B) were fixed with zinc fixative, which works well for CD90 and ABCG2 antibodies. An image from both low magnification and high magnification in each situation is shown. C: Only a small subset of cancer cells express survivin. Human breast cancer (upper panel) and mouse carcinoma (middle panel) stained with survivin rabbit mAb (#2808, Cell Signaling). Mouse carcinoma stained with normal rabbit IgG.