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.
We investigate the relationship between the fate and healing effect of transplanted mesenchymal stromal cells (MSCs) in a rat diabetic skin wound model. Rats are treated with streptozotocin to induce diabetic conditions. A full-thickness skin defect is surgically made on the head of diabetic rats, and covered with an artificial dermis impregnated with either bone marrow cells (BMCs) or bone-marrow-derived MSCs from firefly luciferase (luc) transgenic (Tg) rats. Wound healing is evaluated using planimetry and immunohistochemistry, and the fate of transplanted MSCs is determined using in-vivo luminescent imaging. The diabetic wound treated with MSCs-impregnated artificial dermis is significantly smaller than that treated with artificial dermis alone at 1 week postoperation. Photons of luc+ MSCs are detected at the transplanted site during healing (3 weeks), whereas those of luc+ MSCs are depleted only after 1 week postimplantation. Immunohistochemistry at the healing site treated with MSCs demonstrates that CD31+ vessels increase with expression of vascular endothelial growth factor, suggesting that MSCs accelerate angiogenesis. These findings suggest that transplanted MSCs could be retained at wound sites during the healing process in a diabetic rat model, and subsequently promote wound healing through angiogenesis.
KEYWORDS: Bone, Tissues, In vivo imaging, Cartilage, Stem cells, Green fluorescent protein, Luminescence, Regenerative medicine, Transplantation, Imaging systems
Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine because they can be harvested in
a relatively less invasive manner, easily isolated, and expanded with multipotentiality. Bone marrow seems to be the
most commonly used tissue as a source for MSCs at present. However, there are emerging reports to describe that MSCs
exist in most mesenchymal tissues. We have recently compared in vivo chondrogenic potential in MSCs derived from
various mesenchymal tissues and demonstrated that synovium-MSCs and bone marrow-MSCs possessed greater
chondrogenic ability than other mesenchymal tissue-derived MSCs. This indicates that those MSCs are promising
cellular sources for cartilage regeneration. As the fate of synovium-MSCs is unclear after transplantation, we herein
established MSCs using double transgenic rats expressing either Luciferase/GFP or Luciferase/LacZ. The cellular fate of
MSCs could be traced by an in vivo luciferase-based luminescent imaging system, and also followed histologically by
green fluorescence and by X-gal staining, respectively. Thus, both synovium-MSCs and bone marrow-MSCs expressing
Luciferase/GFP or Luciferase/LacZ provide powerful tools not only for cell tracking in vivo but also for histological
analysis, leading to a compelling experimental model of cartilage regeneration with cell therapy.
KEYWORDS: Bone, In vivo imaging, Luminescence, Green fluorescent protein, Transplantation, Blood, Imaging systems, Stem cells, Visualization, Animal model studies
Because bone marrow is an adequate site for bone marrow stem cells, intra-bone marrow - bone marrow transplantation
(IBM-BMT) is an efficient strategy for bone marrow transplantation (BMT). However, the fate of the transplanted cells
remains unclear. Herein, we established a dual-colored transgenic rat system utilizing green fluorescent protein (GFP)
and a luciferase (luc) marker. We then utilized this system to investigate the in vivo kinetics of transplanted bone marrow
cells (BMCs) after authentic intravenous (IV)-BMT or IBM-BMT. The in vivo fate of the transplanted cells was tracked
using an in vivo luminescent imaging technique; alterations in peripheral blood chimerism were also followed using flow
cytometry. IBM-BMT and IV-BMT were performed using syngeneic and allogeneic rat combinations. While no
difference in the proliferation pattern was observed between the two treatment groups at 7 days after BMT, different
distribution patterns were clearly observed during the early phase. In the IBM-BMT-treated rats, the transplanted BMCs
were engrafted immediately at the site of the injected bone marrow and expanded more rapidly than in the
IV-BMT-treated rats during this phase. Graft-versus-host disease was also visualized. Our bio-imaging system using
dual-colored transgenic rats is a powerful tool for performing quantitative and morphological assessments in vivo.
In vivo imaging strategies provide cellular and molecular events in real time that helps us to understand biological processes in living animals. The development of molecular tags such as green fluorescent proteins and luciferase from the firefly Photinus pyralis has lead to a revolution in the visualization of complex biochemical processes. We developed a novel inbred transgenic rat strain containing firefly luciferase based on the transgenic (Tg) technique in rats. This Tg rat expressed the luciferase gene ubiquitously under control of the ROSA26 promoter. Cellular immune responsiveness against the luciferase protein was evaluated using conventional skin grafting and resulted in the long-term acceptance of Tg rat skin on wild-type rats. Strikingly, organ transplant with heart and small bowel demonstrated organ viability and graft survival, suggesting that cells from luciferase-Tg are transplantable to track their fate. Taking advantage of the less immunogenic luciferase, we also tested the role of hepatocyte-infusion in a liver injury model, and bone marrow-derived cells in a skin defect model. Employed in conjunction with modern advances in optical imaging, this luciferase-Tg rat system provides an innovative animal tool and a new means of facilitating biomedical research such as in the case of regeneration medicine.
We developed the Alb-DsRed2 transgenic (Tg) rat designed with liver-specific expression of the red fluorescent protein, DsRed2. Herein, we report high expression of DsRed2 in neonate liver of both sexes, although they were sexually dimorphic and exhibited a male-specific pattern in adult rats. In an effort to examine the expression in each animal under development, we employed an in vivo Bio-imaging system to quantitatively estimate hepatic DsRed2 expression levels. The temporal profiles pertaining to DsRed expression were similar in male and female Tg rats until 28 days old. The levels in both sexes decreased gradually following birth, and were not detectable at 21 days. Subsequently, expression in males increased again at 35 days and was maintained at a persistently high level thereafter. On the other hand, expression in females disappeared steadily. Although hepatic DsRed expression levels in gonadectomized Tg rats was not significantly different, DsRed expression in hypophysectomized female Tg rats appeared dramatically 72 hr following operation. Hepatocytes were collected from adult Tg rats and cultured in conditioning medium. DsRed expression in female hepatocytes could be detected 72 hr following culturing. These results suggest that hepatic DsRed expression in female rats is regulated in vivo by the pituitary. This report is shows use of Alb-DsRed2 Tg rats in conjunction with a novel bio-imaging system represents a powerful experimental system.
KEYWORDS: Green fluorescent protein, Skin, Ultrasonography, Imaging systems, Medicine, Tolerancing, Transplantation, Visualization, Health sciences, Medical research
GFP is a fluorescent product of the jellyfish Aequorea victoria and has been used for a variety of biological experiments as a reporter molecule. While GFP possesses advantages for the non-invasive imaging of viable cells, GFP-positive cells are still considered potential xeno-antigens. It is difficult to observe the precise fate of transplanted cells/organs in recipients without immunological control. The aim of this study was to determine whether intrathymic injection of GFP to recipients and the depletion of peripheral lymphocytes could lead to donor-specific unresponsiveness to GFP-expressed cell. LEW rats were administered intraperitoneally with 0.2 ml of anti-rat lymphocyte serum (ALS) 1 day prior to intrathymic injection of donor splenocytes or adeno-GFP vector. Donor cells and vector were non-invasively inoculated into the thymus under high frequency ultrasound imaging using an echo-guide. All animals subsequently received a 7 days GFP-expressed skin graft from the same genetic background GFP LEW transgenic rat. Skin graft survival was greater in rats injected with donor splenocytes (23.6+/-9.1) compared with adeno-GFP (13.0+/-3.7) or untreated control rats (9.5+/-1.0). Intrathymic injection of donor antigen into adult rats can induce donor-specific unresponsiveness. Donor cells can be observed for a long-term in recipients with normal immunity using this strategy.
The rat represents an excellent mammalian model for broadening medical knowledge, and a wealth of information on its physiology has been obtained from its use as an experimental organism. Furthermore, its ample body size allows various surgical manipulations that cannot be performed on a mouse. Many rat models mimic human diseases and have therefore been used in a variety of biomedical studies, including physiology, pharmacology, and transplantation. In an effort to create specifically designed rats for new biomedical research and the field of regenerative medicine, we develop an engineered rat system on the basis of transgenic technology and succeed in establishing unique rats that possess genetically encoded color probes: green fluorescent protein (GFP), DsRed2 (red liver), Cre/LoxP (red to green), and LacZ (blue and luminescence). In this work, we highlight their characteristics and describe recent applications for tissue engineering and regeneration. Coupled with recent progress in modern imaging systems, these transgenic rats are providing powerful tools for the elucidation of many cellular processes in biomedical science, and may lead to innovative medical treatments.
The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats’ system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.
The genetic modification for organ transplantation is one of the most promising strategies to regulate allogeneic immune response. Organ-selective gene transfer has especially benefit to control local immune responses. Based on the catheter technique, we tested to deliver naked plasmid DNA to target graft organs of rats (liver and limbs) by a rapid injection (hydrodynamics-based transfection). Recent advances in transplantation have been achieved by visualization of cellular process and delivered gene expression during the inflammatory process by using non-invasive in vivo imaging. Herein, we examined the fate of genetically modified grafts using a firefly luciferase expression plasmid. For liver modification before transplantation, 6.25% of body weight PBS containing plasmid DNA was injected into the liver through the inferior vena cava using a catheter, and the liver was subsequently transplanted to the recipient rat. For limb modification, the femoral caudal epigastric vein was used. In the rat liver transplantation model, substantial luciferase expression was visualized and sustained for only a few days in the grafted liver. We also addressed stress responses by this hydrodynamics procedure using reporter plasmids containing cis-acting enhancer binding site such as NF-kappa B, cAMP, or heat shock response element. In contrast to hepatic transduction, this genetic limb targeting achieved long lasting luciferase expression in the muscle for 2 months or more. Thus, our results suggest that this catheter-based in vivo transfection technique provides an effective strategy for organ-selective gene modification in transplantation, and the bioluminescent imaging is broadening its potential for evaluation to various preclinical studies.
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