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Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation. (C) 2009 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3241985]

Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken P-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.

Background. The characteristics of adenovirus-mediated gene transfer into the kidney are not well examined. We studied the effects of contact time and temperature on adenovirus-mediated transgene expression in rat kidneys, using catheter-based in vivo gene transfer and a rat renal transplant model ex vivo. Methods. An adenovirus vector containing the luciferase (Ad-Luc) or beta-galactosidase (Ad-LacZ) gene was introduced in vivo into the kidney via a renal artery catheter. Various contact times and temperatures were evaluated. Ex vivo, the renal graft was injected with Ad-Luc through the renal artery, chilled for 60 min and then transplanted. Luciferase expression was evaluated periodically by a non-invasive bioimaging system or histology. Cells expressing the LacZ gene were identified by immunoelectron microscopy. Results. In in vivo gene transfer, successful transgene expression was achieved; however, its efficiency was independent of contact time or temperature. In ex vivo gene transfer, transgene expression in the renal graft peaked early and gradually decreased. Strong gene expression was observed in the recipients' livers. LacZ expression was detected in fibroblasts, parietal epithelial cells of Bowman's capsule, mesangial cells, podocytes and tubular cells. Conclusions. This study generated new information about in vivo and ex vivo gene transfer into the kidney, which would be useful for renal gene therapy.

Rodent and human clinical studies have shown that transplantation of bone marrow stem cells to the ischemic myocardium results in improved cardiac function. In this study, cynomolgus monkey acute myocardial infarction was generated by ligating the left anterior descending artery, and autologous CD34(+) cells were transplanted to the peri-ischemic zone. To track the in vivo fate of transplanted cells, CD34(+) cells were genetically marked with green fluorescent protein (GFP) using a lentivirus vector before transplantation (marking efficiency, 41% on average). The group receiving cells (n = 4) demonstrated improved regional blood flow and cardiac function compared with the saline-treated group (n =4) at 2 weeks after transplant. However, very few transplanted cell-derived, GFP-positive cells were found incorporated into the vascular structure, and GFP-positive cardiomyocytes were not detected in the repaired tissue. On the other hand, cultured CD34(+) cells were found to secrete vascular endothelial growth factor (VEGF), and the in vivo regional VEGF levels showed a significant increase after the transplantation. These results suggest that the improvement is not the result of generation of transplanted cell-derived endothelial cells or cardiomyocytes; and raise the possibility that angiogenic cytokines secreted from transplanted cells potentiate angiogenic activity of endogenous cells.

Many endogenous gene expressions in the liver are well known to be predominant in males, compared with those of females. In contrast, the fate of hepatic transgene expression between sexes is not fully understood. Here we studied whether sex hormones changed hepatic transgene expression in the ubiquitous CAG promoter-driven lacZ transgenic (Tg) rat. Both sexes of CAG-lacZ Tg rats received gonadectomy. Liver biopsy was taken weekly to determine the change of transgene expression. Histological result of adult males showed mosaic lacZ expression but it was negative in adult females, while livers in neonatal stage showed comparable expression of lacZ. Other organs exhibited equal expression in both sexes. At 2 weeks after castration, lacZ expression in male liver was significantly decreased and became negative after 4 weeks while no significant difference was observed in the lacZ expression pattern in other organs. After ovariectomy, lacZ expression in female liver remained undetectable. Moreover, testosterone treatment to gonadectomized rats of both sexes could enhance lacZ expression in the liver. In summary, we report that CAG-lacZ Tg rats demonstrate sexual dimorphism of transgene expression specifically only in the liver. Testosterone administration mediated upregulation of liver lacZ expression. Our findings suggested that androgen, especially testosterone, plays an important role in the hepatic transgene expression.

Introduction. Recently, human hand transplantation in Europe has shown that motor function may be recovered in some cases. However, little is known about cell trafficking involved the graft nerve. We have succeeded to use green fluorescent protein transgenic (GFPTg) rats with various cells strongly expressing GFP in a model a long-term survival of limb graft. In this model, we found retrograde migration of GFP-positive donor cells through the sciatic nerve anastomosis. It is well known that cellular components in the peripheral nerve graft especially Schwann cells, play an important role in the axonal regeneration promoted by nerve grafting. However, it was difficult to distinguish the cellular component of the nerve graft from recipient cells. The purpose of this study was to evaluate the migration of donor origin cells to the recipient's nerve and to examine the contribution of these cells in axonal regeneration using a simplified model of sciatic grafting. Methods. Nerve defects were created in recipient rats, using three experimental combinations: group 1: wild-type rats from GFP Tg rats; group 2: GFP Tg rats from wild-type rats; group 3: wild-type rats from GFP Tg rats whose nerve grafts had been pretreated by freeze-thawing cycles (representing an acellular graft). The sciatic nerve specimens were examined under excitation light at 1, 2, and 3 weeks after transplantation. Results. GFP-positive area expanded clearly beyond the anastomosis both proximally and distally in group 1 and infiltrated into the middle of the null graft in group 2. On the contrary, freeze-thawing grafts donated GFP Tg rats lost GFP expression completely. Columns of GFP-positive cells were formed in the degenerated graft migrated into the recipient's nerve both ante- and retrograde. The S100-positive GFP-positive cells were considered to be graft-origin Schwarm cells. The regenerating axons were accompanied with these double-positive cells in the recipient nerve. In conclusion, we have visualized the contribution of graft cells to axonal regeneration beyond a peripheral nerve anastomosis.

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

Background. Glomerular crescent formation is a prominent feature of aggressive forms of glomerulonephritis (GN) and is associated with a poor prognosis. We investigated whether the potent immunosuppressive agent mycophenolate mofetil (MMF) could prevent crescent formation in a model of antiglomerular basement membrane (GBM) GN in the rat. Methods. GN with glomerular crescents was induced by the injection of anti-GBM antibody to female Wistar-Kyoto (WKY/NCrj) rats. The experimental rats were divided into two groups: rats received vehicle (0.5% carboxymethy1cerlose) or MMF (20 mg/kg/day) orally. Body weight was measured and the urine and blood samples were evaluated. The rats were sacrificed at day 14, and histological analysis was performed. The mRNA expression of cytokines and adhesion molecules in the kidney was analysed by reverse transcription polymerase chain reaction (RT-PCR). Results. Marked proteinuria, glomerular crescent formation and glomerulosclerosis were observed in this model, and these were significantly reduced by MMF treatment. Marked glomerular macrophage and T-cell infiltration was also observed, and MMF treatment significantly inhibited macrophage but not T-cell infiltration. RT-PCR and immunohistochemical analysis revealed that mRNA and protein expression of osteopontin was decreased by the treatment with MMF. In addition, MMF treatment in the early stages of GN could inhibit proteinuria, glomerular crescent formation and glomerulosclerosis. Conclusions. These findings suggest therapeutic potential for MMF in the inhibition of glomerular crescent formation in GN and provide new insights into the mechanism underlying the amelioration of crescentic GN by MMF treatment.

Background. Transplant arteriosclerosis is one of the main features of chronic graft failure in organ transplantation. In this article, the authors investigate mechanisms of mycophenolate mofetil (MMF) on prevention of transplant arteriosclerosis in a rat aortic allograft model. Methods. Orthotopic rat abdominal aortic transplantation was performed from Brown Norway (RT1(n)) to Lewis (RT1(1)) rats. The recipients were divided into three oral treatment groups: (1) vehicle; (2) MMF40 (40 mg/kg); and (3) MMF20 (20 mg/kg). The authors histologically and immunohistochemically evaluated neo-intima formation; infiltration of macrophages and T cells; and expression of endothelin (ET)-1, platelet-derived growth factor (PDGF)-B, PDGF receptor-beta (Rbeta), transforming growth factor (TGF) beta1, and osteopontin (OPN). Using cultured rat vascular smooth muscle cells (VSMC), effects of mycophenolic acid (MPA) on ET-1-induced proliferation and ERK1/2 activation were also examined in vitro. Results. In the vehicle group, marked neointima formation was observed, with massive macrophages and T-cell infiltration in neointima, media, and adventitia. Marked expression of ET-1, PDGF-B, PDGFR-beta, TGFbeta1, and OPN were also observed in neointima. In the MMF40 and MMF20 groups, neointima formation was halted, but macrophages and T cells were infiltrated in the adventitia and adhered to the endothelium. In the MMF40 group, medial infiltration by macrophages and T cells and intimal expression of ET-1, PDGF-B, PDGFR-beta, TGFbeta1, and OPN was inhibited compared with the vehicle and MMF20 groups. Furthermore, MPA inhibited ET-1-induced VSMC proliferation but failed to inhibit its ERK1/2 activation. Conclusions. MMF treatment might have preventive potential in transplant patients with chronic vasculopathy through inhibition of VSMC proliferation.

Background Embryonic stem (ES) cells continually proliferate and can generate large numbers of differentiated cells. Genetic manipulation of transplantable cells derived from primate ES cells offers considerable potential for development research and regenerative cell therapy. However, protocols for efficient gene transfer into primate ES-cell-derived cells have not yet been established. Methods Spontaneously contracting areas were derived from cynomolgus monkey ES cells. Features of cardiomyocytes in the area were analyzed according to gene expression (RT-PCR), morphology (immunostaining and electron microscopy), and function (intracellular calcium transience). Beating cells were transduced using a simian immunodeficiency virus (STV) vector expressing enhanced green fluorescence protein (EGFP), then transplanted into ischemic rat myocardium. Results Beating cells derived from monkey ES cells displayed gene expression, ultrastructural and functional properties of early-stage cardiomyocytes. Highly efficient (97% cardiac phenotype) and stable transduction of these ES-cell-derived cardiomyocytes was achieved using SIV vector without altering contractile function. In addition, transduced cardiomyocytes survived in the myocardium of a rat myocardial infarction model. Conclusions A lentiviral vector system based on SIV represents a useful vehicle for genetic modification of cardiomyocytes derived from primate ES cells, and can extend the application of primate ES cells to gene therapy. Copyright (C) 2003 John Wiley Sons, Ltd.