Murine cytomegalovirus immediate-early promoter directs astrocyte-specific expression in transgenic mice

Abstract

Murine cytomegalovirus (MCMV), which causes acute, latent, and persistent infection of the natural host, is used as an animal model of human cytomegalovirus (HCMV) infection. Transcription of MCMV immediate-early (IE) genes is required for expression of the early and late genes and is dependent on host cell transcription factors. Cell-type-specific expression activity of the MCMV IE promoter was analyzed in transgenic mice generated with the major IE (MIE) enhancer/promoter involving nucleotides -1343 to -6 (1338 bp) connected to the reporter gene lacZ. Distinct expression was observed in the brain, kidneys, stomach, and skeletal muscles. Weak expression was observed in a portion of the parenchymal cells of the salivary glands and pancreas, and expression was hardly detected in the lungs, intestine, or immune and hematopoietic organs such as the thymus, spleen, lymph nodes, and bone marrow. The spectrum of organs positive for expression was narrower than that of the HCMV MIE promoter-lacZ transgenic mice reported previously and showed a greater degree of cell-type specificity. Interestingly, astrocyte-specific expression of the transgene was observed in the brain and primary glial cultures from the transgenic mice by combination of beta-galactosidase (beta-Gal) expression and immunostaining for cell markers. However, the transgene was not expressed in neurons, oligodendroglia, microglia, or endothelial cells. Furthermore, the beta-Gal expression in glial cultures was stimulated significantly by MCMV infection or by addition of calcium ionophore. These observations indicated that expression activity of the MCMV IE promoter is strictly cell-type specific, especially astrocyte-specific in the brain. This specific pattern of activity is similar to that of natural HCMV infection in humans.

Figure 1.

A: Schema of the MCMV-MIEpro1-lacZ transgene DNA fragment (transgene; 5.1 kb) used to generate transgenic mice. The MCMV major IE promoter (MIEpro1) (nucleotides −1343 to −6) was inserted into the pnlacF vector. N, nuclear localization signal. Splice and polyadenylation signals were from the mouse protamine (mP1) gene. B: Screening of integrated transgene DNA by multiplex PCR. DNA from the tails of mice was tested for the presence of the transgene and host gene by PCR using specific primers (primer A and B) and the mouse apoE internal control primers, respectively. PCR amplification was performed, and the reaction products were run on 2% agarose gels, which were then stained with ethidium bromide. Lanes 10 to 13 show 0, 0.1, 1, and 10 copies of the MCMV-MIEpro1-lacZ plasmid, respectively. C: Southern blot of the tail DNA from the transgenic mice. Aliquots of 15 μg of tail DNA were digested with BamHI, run in 0.9% agarose gels, transferred on nylon membranes, and probed with ³³P-labeled BamHI fragment (1.3 kb; A). Control lanes show 10 copies (lane 1), 1 copy (lane 2), and 0 copies (lane 3) of MCMV-MIEpro1-lacZ with 15 μg of normal mouse tail DNA. Lane 4, Tg-1; lane 5, Tg-2; lane 6, Tg-3.

Figure 2.

Immunohistochemical staining of expression of the MCMV MIEpro1-lacZ transgenic mice. Transgenic mice (Tg-1) generated by the MCMV-MIEpro1-lacZ transgene were examined by immunohistochemical staining. Tissues from 2-month-old Tg-1 mice were fixed in 4% PFA and embedded in paraffin. Deparaffinized sections were reacted with the anti-β-galactosidase antibody (anti-β-Gal), followed by goat anti-rabbit IgG horseradish peroxidase (HRP), and then colored with 3-amino-9-ethyl carbazole (AEC). A: Cerebral cortex (arrow, β-Gal-positive cells); B: Hippocampus (pcl, pyramidal cell layer); C: Cerebellum (m, molecular layer; gc, granular cells; arrows, β-Gal-positive cells (Bergmann glia); arrowheads, Purkinje cells); D: Kidney (g, glomerulus; pt, proximal tubules; dt, distal tubules); E: Salivary gland (arrow, slight β-Gal-positive cells); F: Parenchymal cells around the central vein of the liver (cv, central vein); G: Gastric mucosa; H: Small-intestinal mucosa (arrow, β-Gal-positive cells); I: Spleen.

Figure 3.

Combination of X-Gal staining and immunohistochemical staining. A: Slices of the cerebral cortex of the Tg-1 mice were first subjected to the X-Gal reaction and then embedded in paraffin. Deparaffinized sections were reacted with anti-NSE Ab and colored with AEC. B: Section from the same slice with X-Gal reaction but immunostained with anti-GFAP Ab . C: Fibrous astrocytes in the white matter around the hippocampus. pcl, pyramidal cell layer. D to F: Primary glial culture from the cerebral cortex of 3-day-old Tg-1 mice. After X-gal staining, the cells were stained with the GFAP Ab (D), with F4/80 Ab (microglia/macrophages; E), or with MAG Ab for oligodendroglia (F).

Figure 4.

Chemiluminescent assay for β-galactosidase of glial cultures from the transgenic mice. A: Glial cells plated in 12-well plates were infected or mock-infected with MCMV (Smith strain) and assayed 1 to 12 hours after infection. B: Glial cells were added with calcium ionophore (A23187) at a concentration of 5 mol/L or not added and cultured until 12 hours.