The murine stem cell virus promoter drives correlated transgene expression in the leukocytes and cerebellar Purkinje cells of transgenic mice.

The murine stem cell virus (MSCV) promoter exhibits activity in mouse hematopoietic cells and embryonic stem cells. We generated transgenic mice that expressed enhanced green fluorescent protein (GFP) under the control of the MSCV promoter. We obtained 12 transgenic founder mice through 2 independent experiments and found that the bodies of 9 of the founder neonates emitted different levels of GFP fluorescence. Flow cytometric analysis of circulating leukocytes revealed that the frequency of GFP-labeled leukocytes among white blood cells ranged from 1.6% to 47.5% across the 12 transgenic mice. The bodies of 9 founder transgenic mice showed various levels of GFP expression. GFP fluorescence was consistently observed in the cerebellum, with faint or almost no fluorescence in other brain regions. In the cerebellum, 10 founders exhibited GFP expression in Purkinje cells at frequencies of 3% to 76%. Of these, 4 mice showed Purkinje cell-specific expression, while 4 and 2 mice expressed GFP in the Bergmann glia and endothelial cells, respectively. The intensity of the GFP fluorescence in the body was relative to the proportion of GFP-positive leukocytes. Moreover, the frequency of the GFP-expressing leukocytes was significantly correlated with the frequency of GFP-expressing Purkinje cells. These results suggest that the MSCV promoter is useful for preferentially expressing a transgene in Purkinje cells. In addition, the proportion of transduced leukocytes in the peripheral circulation reflects the expression level of the transgene in Purkinje cells, which can be used as a way to monitor transgene expression properties in the cerebellum without invasive techniques

GFP expression in the bodies and leukocytes of MSCV-GFP founder mice.

<p>(A–D) Twelve founder pups were obtained from 2 independent experiments (A, B) and (C, D). Brightfield (B, D) and GFP fluorescence images (A, C) of the transgenic pups were obtained using a fluorescence stereoscopic microscope. (E–H) Robust GFP expression was observed in the leukocytes (arrow) and platelets (arrowhead) of a representative MSCV-GFP mouse (ID0Tg-7, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051015#pone-0051015-t001" target="_blank">Table 1</a>) (E, F) but not in wild-type mice (G, H). (I) Immunolabeling of GFP-positive leukocytes (arrow) but not GFP-positive platelet (arrowhead) for CD45. Peripheral blood cells from a F1 MSCV-GFP mouse (IE1Tg-2, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051015#pone-0051015-t001" target="_blank">Table 1</a>) were immunolabeled for CD45. (J, K) Flow cytometric analysis of leukocytes in the MSCV-GFP mouse. Peripheral blood cells from a F1 transgenic mouse (IE1Tg-2) and a wild-type mouse were immunolabeled for CD45 following hemolysis of erythrocytes. Leukocytes were analyzed using a flow cytometer and CellQuest pro software. Scale bar, 10 µm.</p

Expression of GFP during the development of C57BL/6 MSCV-GFP mice.

<p>(A) No GFP expression was observed in the Purkinje cells at P5. Cerebellar sections obtained from MSCV-GFP mice at P5 (left), P10 (middle) and P30 (right) were immunolabeled for calbindin. Images of native GFP fluorescence (upper), calbindin immunoreactivity (middle) and merged images (lower) are presented. (B) Brightfield (upper) and fluorescent (lower) images of MSCV-GFP mice at P5, P10 and P30. Weak, but clearly detectable, levels of GFP were observed in the thymus from as early as P5 and increased thereafter. GFP fluorescence in the skeletal muscles became overt at P10. T; thymus, H; heart, Lu; lung, Li; liver, I; intestine, St; stomach, Sp; spleen.</p

Properties of MSCV-GFP founder and F1 mice in comparison to wild-type mice.

<p>The first letter (I) of the identification (ID) indicates that the mouse was generated on an ICR background. Littermates have the same letter (A–E) at the second position. Numbers (0 or 1) at the third position show the filial generation of the transgenic mice and are followed by an abbreviation of the genotype, either wild-type (Wt) or transgenic (Tg). The final numbers are serial numbers in the same filial generation. Since there was no wild type in the F0 generation and also in the F1 offspring, the wild-type ICR mice (IAWt-1, IAWt-2 and IBWt-3) were obtained from a same supplier. F; female, M.</p

GFP expression in the body and leukocytes of a C57BL/6 MSCV-GFP mouse.

<p>(A, B) GFP expression in circulating leukocytes (arrow) and platelets (arrowhead) in a transgenic mouse (A) but not in a wild-type littermate (B). (C–F) Flow cytometric analysis shows that almost all of the circulating leukocytes in the transgenic mice (C, D) have higher GFP fluorescence levels than those of wild-type mice (E, F). (G) Illumination of a F1 transgenic mouse pup (Tg) at 3 days of age, together with a wild-type littermate (WT). (H) Southern blot analysis of the blood of the founder mouse (F0), 15 F1 pups (F1) and a wild-type littermate (WT). Upper illustration shows the genomic region (450 bp) where the DIG-labeled probe bound. The lane marker (M) on the left of the gel represents base pairs. Scale bar, 20 µm.</p

Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia

Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin–proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases

Expression of GFP in the lymphoid tissues and myocytes of C57BL/6 MSCV-GFP mice.

<p>(A, B) Brightfield (left) and fluorescent (right) images of an MSCV-GFP mouse (A) and a wild-type littermate (B). The brightest GFP fluorescence was observed in the thymus, followed by the skeletal muscle. T; thymus, H; heart, Lu; lung, Li; liver, I; intestine, Sp; spleen. (C, D) Histological sections of the thymus (C) and skeletal muscle (D) immunolabeled for GFP (green) and CD45 (C, red) or a-actinin (D, red). The upper and lower images were obtained from an MSCV-GFP mouse and a wild-type littermate, respectively. (E–H) Native GFP fluorescence images from Peyer’s patch of the intestine (E), peripheral lymph node (F), spleen (G) and bone marrow (H) of an MSCV-GFP mouse. No fluorescence signal was detected in the same regions of wild-type littermates. Scale bars, 50 µm (C), 20 µm (D), 100 µm (E), 50 µm (F) and 20 µm (G, H).</p

MSCV promoter-mediated expression of GFP in various types of cultured cells.

<p>(A) Lentiviral vectors express enhanced green fluorescent protein (GFP) under the control of the MSCV promoter. To remove TAT-dependent transcription, the U3 region of the LTR was deleted. The Δgag (deleted gag sequence) was included to enhance packaging efficiency. For improved viral-mediated gene transfer, the central polypurine tract (cPPT) and central termination sequence (CTS) were added in Δpol (deleted pol sequence). RRE; Rev response element. (B) Expression of GFP in mouse embryos. (C) Expression of GFP in human embryonic kidney (HEK) 293T cells. (D) Cerebellar neuronal culture infected with lentiviral vectors at 1 day in vitro (DIV) and immunolabeled for neuron-specific nuclear protein (NeuN) at DIV 15. In addition to Purkinje cells (arrows), GFP was expressed in granule neurons (arrowhead, inset) and in glia (arrow, inset). Scale bars, 20 µm (B), 25 µm (C), 50 µm (D).</p

Significant correlation between the frequencies of GFP-positive leukocytes and GFP-expressing Purkinje cells.

<p>(A–C) GFP fluorescence of peripheral circulating leukocytes was analyzed by flow cytometry. (D–F) Sagittal sections of the cerebellar vermis from the same animals (A–C) are shown. The results were obtained from IC0Tg-3 (A, D), IC0Tg-1 (B, E) and IC0Tg-6 (C, F) mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051015#pone-0051015-t001" target="_blank">Table 1</a>). Scale bar, 20 µm. (G) The frequency of GFP-expressing leukocytes (% GFP(+) leukocytes) obtained from 12 MSCV-GFP founder mice was plotted against the frequency of GFP-expressing Purkinje cells (% GFP(+) Purkinje cells). The correlation coefficient (r) was 0.84, indicating a significant correlation between the two values.</p