Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.
Animals ; Green Fluorescent Proteins ; Lentivirus ; Male ; Rats ; Rats, Transgenic ; Spermatogonia/metabolism*

The clinical spectrum of spondyloarthritis is included various diagnostic entities that share clinical, genetic and pathological characteristics. As human tissue specimens of the sacroiliac joints are very difficult to obtain, most of the new concepts have emerged from different animal models of disease. Animal models are available for the study of several different aspects of spondyloarthritis. The models include human leukocyte antigen (HLA) B-27 based on transgenic rat and mouse models, inflammation-driven models, and models of ankylosing enthesitis. Areas of investigation to which these models contribute include the role of HLA B-27, process of spinal and peripheral joint inflammation and calcification, immune responses to candidate antigens and the role of tumor necrosis factor.
Animals ; Humans ; Inflammation ; Joints ; Leukocytes ; Mice ; Models, Animal ; Rats, Transgenic ; Sacroiliac Joint ; Tumor Necrosis Factor-alpha

Human ITP clinical trials have been performed based on observations of clinical uses of drugs, other than on mechanism studies in preclinical models, many studies were stopped because of unexpected complications. So there was need to develop ideal animal model of ITP to improve the understanding of the pathophysiology and the mechanisms of action of therapeutics. In this review, the history of animal models of ITP was retrospected, two modeling methods inducing passive and active models, especially transgenic rice model which can more accurately represent human disease pathophysiology, and their application in study of ITP were summarized. An animal model for human immune thrombocytopenia allows detailed studies of the mechanism, kinetics, and therapy of human immune thrombocytopenia.
Animals ; Disease Models, Animal ; Mice ; Mice, Transgenic ; Purpura, Thrombocytopenic, Idiopathic ; Rats

OBJECTIVETo trace the embryonic stem (ES) cells transplanted into rat brain by labeling the cells with green fluorescent protein (GFP) and by mouse neuronal specific antibody Thy-1 and compare their features.

METHODSFor GFP labeling,transfect pEGFP-N1 plasmid containing GFP and anti-neomycin sequences into embryonic stem cell and add neomycin for more than 10 passages. To test the GFP expression in vivo, the GFP-ES was transplanted into healthy rat brain, and the frozen sectioned slides were observed under fluorescence microscope and laser con-focal microscope 21 days later. For the antibody labeling,embryonic stem cells were directly transplanted into the rat brain. The specific mouse thy-1 antibody was used in immunostaining of transplanted cells. For both of the two labeling method, the slides were also examined by double labeling with the antibodies,neuronal nuclei (NeuN) or glial fibrillary acidic protein (GFAP) to identify the differentiation of transplanted cells.

RESULTSBoth single ES cell and cell pellets expressed bright green fluorescence the day after plasmid transfection, and more than 30% ES cells were labeled. The GFP-labeled cells could still be found gathered around the infusion channel at least 21 days later, but the GFP fluorescent could not be overlapped with NeuN or GFAP staining. On the contrary, Thy-1 antibody overlapped well with NeuN or GFAP staining.

CONCLUSIONSLiposome-helped plasmid GPF transfection is effective in labeling mouse embryonic stem cell in vivo,but is not effective in showing the differentiated cells. On the contrary, Thy-1 antibody can not only show the transplanted cells, but also trace the transplanted cells after their differentiation.

Animals ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; physiology ; transplantation ; Green Fluorescent Proteins ; Male ; Mice ; Mice, Transgenic ; Rats ; Rats, Sprague-Dawley ; Staining and Labeling ; methods

OBJECTIVETo investigate the effect of herpes simplex virus-thymidine kinase (HSV-TK) gene transduced into T cell clone on the treatment of ulcerative colitis (UC) in rat.

METHODSThe T cell clone transduced with HSV-tk was infused into 27 rats with UC. Changes of stimulation index (SI), CD4(+), CD8(+), IL-13, IL-4 were detected, and pathological changes before and after infusion was compared.

RESULTSIn the second and third day after tk+ T clone infusion, the inflammation of colon was assimilated. Two weeks later, the colon began to renovate and mend. The average SI was 7.39+/-1.24 before infusion, and 2.67+/-0.87 after infusion (P<0.05). Peripheral blood levels of CD4(+), CD8(+) or IL-13 and IL-4 in therapeutic group were significantly decreased as compared to control group (P<0.05).

CONCLUSIONHSV-tk gene transduced into T lymphocyte clone and infused back is effective for UC gene therapy and target design in rat.

Animals ; Colitis, Ulcerative ; therapy ; Genes, Transgenic, Suicide ; Genetic Therapy ; Male ; Rats ; Rats, Wistar ; Simplexvirus ; genetics ; T-Lymphocytes ; Thymidine Kinase ; genetics ; Viral Proteins ; genetics

OBJECTIVETo isolate, culture, and identify bone marrow mesenchymal stem cells (BMSCs) from enhanced green fluorescent protein (EGFP)-transgenic rats in vitro.

METHODSBone marrows were isolated from tibia and femur of healthy EGFP-transgenic rats of specific pathogen free (SPF) grade. Then,the whole bone marrow adherent method was used for isolation,culture,and purification of BMSCs. The morphological change was noted by continuous observation under inverted fluorescence microscope. The growth curve of cells was drawn through the method of CCK-8 and the proliferation compared with wild type BMSCs. The surface markers of BMSCs were detected by flow cytometry. The BMSCs were induced to differentiate into osteoblasts, adipocytes, and chondrocytes lineages. The EGFP-BMSCs were transplanted into the rats intravenously, and the expression of GFP was detected.

RESULTSBMSCs stably expressing EGFP gene were obtained successfully, with the fusiform-shaped appearance and the forming of circinate cell colonies. The growth curve of EGFP-MSCs showed the characteristic of active proliferation, showing no significant difference compared with the wild-type BMSCs. The expression rates of the surface markers of BMSCs CD29, CD90, CD34, CD49d, and CD45 were 99.4%, 96.4%, 0.171%, 0.049%, and 0.038%. The GFP were detected in lung 3 days after transplantation. After osteogenic, adipogenic, and chondrogenic induction, oil red-O and alizarin red positive signals and toluidine blue positive cells were detected.

CONCLUSIONSHigh-purity BMSCs stably expressing green fluorescent protein gene can be cultured using the whole bone marrow adherent method. EGFP does not affect the stem cell properties and expresses stably after transplantation. The cells can be used as seed cells for subsequent research.

Adipocytes ; Animals ; Bone Marrow Cells ; Cells, Cultured ; Chondrocytes ; Flow Cytometry ; Green Fluorescent Proteins ; Hematopoietic Stem Cells ; Mesenchymal Stromal Cells ; Osteoblasts ; Rats ; Rats, Transgenic

The transgenic mice that express Cre recombinase in a tissue specific manner is a powerful tool in generating the conditional gene knockout mice. The rat insulin promoter was cloned target the expression of Cre in pancreatic tissue. The Cre gene was modified by adding the nuclear localization signal and the sequence for initiation by eukaryotic ribosomes at 5' terminal of the Cre gene. Cre gene was linked to the intron of human growth factor gene. This construct was introduced into the mouse eggs using microinjection. Seven mice were identified as founders carrying the Cre gene by PCR. The results of RT-PCR showed that the transgenic mouse from one founder could transcribe the foreign gene in pancreas. The Southern blot analysis indicated that the Cre recombinase expressed in pancreas of the transgenic mouse was functional.
Animals ; Blotting, Southern ; Female ; Insulin ; genetics ; Integrases ; genetics ; Mice ; Mice, Transgenic ; Pancreas ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; analysis ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Proteins ; genetics

Aucubin is a small compound naturally found in traditional medicinal herbs with primarily anti-inflammatory and protective effects. In the nervous system, aucubin is reported to be neuroprotective by enhancing neuronal survival and inhibiting apoptotic cell death in cultures and disease models. Our previous data, however, suggest that aucubin facilitates neurite elongation in cultured hippocampal neurons and axonal regrowth in regenerating sciatic nerves. Here, we investigated whether aucubin facilitates the differentiation of neural precursor cells (NPCs) into specific types of neurons. In NPCs cultured primarily from the rat embryonic hippocampus, aucubin significantly elevated the number of GAD65/67 immunoreactive cells and the expression of GAD65/67 proteins was upregulated dramatically by more than three-fold at relatively low concentrations of aucubin (0.01 µM to 10 µM). The expression of both NeuN and vGluT1 of NPCs, the markers for neurons and glutamatergic cells, respectively, and the number of vGluT1 immunoreactive cells also increased with higher concentrations of aucubin (1 µM and 10 µM), but the ratio of the increases was largely lower than GAD expression and GAD immunoreactive cells. The GABAergic differentiation of pax6-expressing late NPCs into GABA-producing cells was further supported in cortical NPCs primarily cultured from transgenic mouse brains, which express recombinant GFP under the control of pax6 promoter. The results suggest that aucubin can be developed as a therapeutic candidate for neurodegenerative disorders caused by the loss of inhibitory GABAergic neurons.
Animals ; Axons ; Brain ; Cell Death ; GABAergic Neurons* ; Hippocampus ; Mice ; Mice, Transgenic ; Nervous System ; Neurites ; Neurodegenerative Diseases ; Neurons ; Plants, Medicinal ; Rats ; Sciatic Nerve

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
Animals ; Apoptosis ; Diet ; Endothelial Cells ; Erectile Dysfunction/prevention & control* ; Fibrosis ; Humans ; Hyperhomocysteinemia/complications* ; Male ; Methionine ; Oxidative Stress ; Penis/pathology* ; Rats ; Rats, Sprague-Dawley ; Rats, Transgenic ; Signal Transduction/genetics* ; Tissue Kallikreins/genetics*

BACKGROUND: Several studies have indicated that a nerve injury enhances the expression of the voltage-gated calcium channel alpha2delta1 subunit (Cavalpha2delta1) in sensory neurons and the dorsal spinal cord. This study examined whether NMDA receptor activation is essential for Cavalpha2delta1-mediated tactile allodynia in Cavalpha2delta1 overexpressing transgenic mice and L5/6 spinal nerve ligated rats (SNL). These two models show similar Cavalpha2delta1 upregulation and behavioral hypersensitivity, without and with the presence of other injury factors, respectively. METHODS: The transgenic (TG) mice were generated as described elsewhere (Feng et al., 2000). The left L5/6 spinal nerves in the Harlan Sprague Dawley rats were ligated tightly (SNL) to induce neuropathic pain, as described by Kim et al. (1992). Memantine 2 mg/kg (10 ul) was injected directly into the L5/6 spinal region followed by 10microl saline. Tactile allodynia was tested for any mechanical hypersensitivity. RESULTS: The tactile allodynia in the SNL rats could be reversed by an intrathecal injection of memantine 2 mg/kg at 1.5 hours. The tactile allodynia in the Cavalpha2delta1 over-expressing TG mice could be reversed by an intrathecal injection of memantine 2 mg/kg at 1.5, 2.0 and 2.5 hours. CONCLUSIONS: The behavioral hypersensitivity was similar in the TG mice and nerve injury pain model, supporting the hypothesis that elevated Cavalpha2delta1 mediates similar pathways that underlie the pain states in both models. The selective activation of spinal NMDA receptors plays a key role in mediating the pain states in both the nerve-injury rats and TG mice.
Animals ; Calcium ; Calcium Channels ; Hyperalgesia ; Hypersensitivity ; Injections, Spinal ; Memantine ; Mice ; Mice, Transgenic ; N-Methylaspartate ; Negotiating ; Neuralgia ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; Sensory Receptor Cells ; Spinal Cord ; Spinal Nerves ; Up-Regulation