Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Exp Anim ; 68(3): 361-370, 2019 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-30996149

RESUMEN

In Japan, it is possible to generate chimeric animals from specified embryos by combining animal blastocysts with human pluripotent stem (PS) cells (animal-human PS chimera). However, the production of animal-human PS chimeras has been restricted because of ethical concerns, such as the development of human-like intelligence and formation of humanized gametes in the animals, owing to the contributions of human PS cells to the brain and reproductive organs. To solve these problems, we established a novel blastocyst complementation technology that does not contribute to the gametes or the brain. First, we established GFP-expressing mouse embryonic stem cells (G-mESCs) in which the Prdm14 and Otx2 genes were knocked out and generated chimeric mice by injecting them into PDX-1-deficient blastocysts. The results showed that the G-mESCs did not contribute to the formation of gametes and the brain. Therefore, in the PDX-1-deficient mice complemented by G-mESCs without the Prdm14 and Otx2 genes, the germline was not transmitted to the next generations. This approach could address concerns regarding the development of both human gametes and a human-like brain upon mouse blastocyst complementation using human stem cells.


Asunto(s)
Blastocisto/citología , Diferenciación Celular/fisiología , Técnicas de Cultivo de Embriones/métodos , Transferencia de Embrión/métodos , Células Madre Embrionarias de Ratones/citología , Animales , Encéfalo/fisiología , Femenino , Células Germinativas/fisiología , Japón , Masculino , Ratones , Ratones Endogámicos ICR
2.
J Vet Med Sci ; 80(4): 662-666, 2018 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-29445073

RESUMEN

Expression of peroxisome proliferator-activated receptor (PPAR) α was investigated in adiponectin knockout mice to elucidate the relationship between PPARα and adiponectin deficiency-induced diabetes. Adiponectin knockout (Adp-/-) mice were generated by gene targeting. Glucose tolerance test (GTT), insulin tolerance test (ITT), and organ sampling were performed in Adp-/- mice at the age of 10 weeks. PPARα, insulin, triglyceride, free fatty acid (FFA), and tumor necrosis factor α (TNFα) were analyzed from the sampled organs. Adp-/- mice showed impaired glucose tolerance and insulin resistance. Additionally, PPARα levels were decreased and plasma concentration of triglyceride, FFA and TNFα were increased. These data may indicate that insulin resistance in Adp-/- mice is likely caused by an increase in concentrations of TNFα and FFA via downregulation of PPARα.


Asunto(s)
Adiponectina/genética , Diabetes Mellitus/metabolismo , Regulación hacia Abajo/fisiología , Ácidos Grasos no Esterificados/metabolismo , PPAR alfa/metabolismo , Animales , Diabetes Mellitus/genética , Regulación de la Expresión Génica/fisiología , Intolerancia a la Glucosa , Insulina/sangre , Ratones , Ratones Noqueados , PPAR alfa/genética , Factor de Necrosis Tumoral alfa
3.
Biochem Biophys Res Commun ; 478(3): 1254-60, 2016 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-27545600

RESUMEN

Most in vivo studies on the conversion to insulin-producing cells with AAV carrying PDX1 gene are performed in rodents. However, there is little information regarding Adeno-associated virus (AAV) carrying PDX1 gene transduced to human liver in vivo because accidental death caused by unpredicted factors cannot be denied, such as the hypoglycemic agent troglitazone with hepatic failure. Here we aim to confirm insulin secretion from human liver transduced with AAV carrying PDX1 gene in vivo and any secondary effect using a humanized liver mouse. As the results, AAV2-PG succeeded to improve the hyperglycemia of STZ-induced diabetic humanized liver mice. Then, the analysis of humanized liver mice revealed that the AAV2-PG was more transducible to humanized liver area than to mouse liver area. In conclusion, the humanized liver mouse model could be used to examine AAV transduction of human hepatocytes in vivo and better predict clinical transduction efficiency than nonhumanized mice.


Asunto(s)
Dependovirus/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/terapia , Terapia Genética , Animales , Diabetes Mellitus Experimental/complicaciones , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/terapia , Insulina/metabolismo , Ratones , Ratones Transgénicos , Transactivadores/metabolismo , Transducción Genética
4.
Lab Anim Res ; 31(2): 93-8, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-26155204

RESUMEN

We developed pancreatic and duodenal homeobox1 (Pdx1) knockout mice to improve a compensatory hyperinsulinemia, which was induced by hyperplasia in the ß cells or Langerhans' islands, as the diabetic model mice. For targeting of Pdx1 gene by homologous recombination, ES cells derived from a 129(+Ter) /SvJcl×C57BL/6JJcl hybrid mouse were electroporated and subjected to positive-negative selection with hygromycin B and ganciclovir. As these results, one of the three chimeric mice succeeded to produce the next or F1 generation. Then, the mouse fetuses were extracted from the mother's uterus and analyzed immunohistologically for the existence of a pancreas. The fetuses were analyzed at embryonic day 14.5 (E14.5) because Pdx1 knockout could not alive after birth in this study. Immunohistochemical staining revealed that 10 fetuses out of 26 did not have any PDX1 positive primordium of the pancreas and that the PDX1 expresses in both the interior and exterior regions of intestine. In particular, one the exterior of the intestine PDX1 was expressed in glands that would be expected to form the pancreas. The result of PCR genotyping with extracted DNA from the paraffin sections showed existence of 10 Pdx1-knockout mice and corresponded to results of immunostaining. Thus, we succeeded to establish a Pdx1-knockout (Pdx1 (-/-)) mice.

5.
Transgenic Res ; 20(6): 1245-52, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21359657

RESUMEN

In vitro fertilization (IVF) is a valuable technique for the propagation of experimental animals. IVF has typically been used in mice to rapidly expand breeding colonies and create large numbers of embryos. However, applications of IVF in rat breeding experiments have stalled due to the inconvenient laboratory work schedules imposed by current IVF protocols for this species. Here, we developed a new rat IVF protocol that consists of experimental steps performed during common laboratory working hours. Our protocol can be completed within 12 h by shortening the period of sperm capacitation from 5 to 1 h and the fertilization time from 10 to 8 h in human tubal fluid (HTF) medium. This new protocol generated an excellent birth rate and was applicable not only to closed colony rat strains, such as Wistar, Long-Evans, and Sprague-Dawley (SD), but also to the inbred Lewis strain. Moreover, Wistar and Long-Evans embryos prepared by this protocol were successfully frozen by vitrification and later successfully thawed and resuscitated. This protocol is practical and can be easily adopted by laboratory workers.


Asunto(s)
Embrión de Mamíferos/fisiología , Fertilización In Vitro/métodos , Ciencia de los Animales de Laboratorio/métodos , Animales , Cruzamiento/métodos , Criopreservación/métodos , Medios de Cultivo/química , Trompas Uterinas/fisiología , Femenino , Masculino , Oocitos/fisiología , Ratas , Ratas Endogámicas Lew , Ratas Long-Evans , Ratas Sprague-Dawley , Ratas Wistar , Resucitación/métodos , Especificidad de la Especie , Espermatozoides/fisiología , Factores de Tiempo , Vitrificación
6.
Methods Mol Biol ; 597: 93-108, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20013228

RESUMEN

Transgenic rats with a simple plasmid vector smaller than 20 Kb show insufficient expression and tissue specificity of the introduced transgene. Vectors derived from yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC), consisting of DNA fragments up to approximately 1 Mb (YAC) and approximately 200 Kb (BAC), respectively, and containing various endogenous regulatory sequences, were expected to work well and showed expression profiles comparable to their endogenous counterparts in transgenic animals. While attempting to make transgenic rats using YAC and BAC vectors, we faced two problems: how to prepare sufficiently concentrated intact DNA and how to reliably microinject a large DNA fragment into the fragile pronuclear ova of the rat. After solving these problems, we were able to make transgenic rats by introducing YAC/BAC gene constructs (YACs/BACs) into the pronuclear ova. And then we examined the relative transcription rates of these genes in the transgenic rats. In this chapter, we focus on this injection process.


Asunto(s)
Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Levadura/genética , ADN/administración & dosificación , Microinyecciones/métodos , Ratas Transgénicas/genética , Animales , ADN/genética , Diseño de Equipo , Vectores Genéticos , Microinyecciones/instrumentación , Óvulo/citología , Ratas , Transgenes
7.
Cell Biol Int ; 32(11): 1344-52, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18725308

RESUMEN

A tracheal epithelial cell line RTEC11 was established from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen. The cells grew continuously at a permissive temperature of 33 degrees C but not at a non-permissive temperature of 39 degrees C. Morphological and functional investigations demonstrated that the cells were polarized epithelial cells maintaining a regulated permeability barrier function. Interestingly, the expression levels of Muc1 (mucin 1) and Scgb1a1 (uteroglobin), non-ciliated secretory cell markers, and Tubb4 (tubulin beta 4), a ciliated cell marker, were significantly increased under the cell growth-restricted condition. Global gene expression and computational network analyses demonstrated a significant genetic network associated with cellular development and differentiation in cells cultured at the non-permissive temperature. The tracheal epithelial cell line RTEC11 with unique characteristics should be useful as an in vitro model for studies of the physiological functions and gene expression of tracheal epithelial cells.


Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Células Epiteliales/metabolismo , Temperatura , Tráquea/metabolismo , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Proteínas Portadoras/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/genética , Proliferación Celular , Cilios/metabolismo , Simulación por Computador , Células Epiteliales/citología , Regulación de la Expresión Génica/genética , Modelos Biológicos , Mucina-1/metabolismo , Redes Neurales de la Computación , Ratas , Ratas Transgénicas , Tráquea/citología , Tubulina (Proteína)/metabolismo , Uteroglobina
8.
J Cell Physiol ; 217(2): 388-99, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18543246

RESUMEN

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.


Asunto(s)
Barrera Hematonerviosa/metabolismo , Permeabilidad Capilar , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Pericitos/metabolismo , Nervio Ciático/irrigación sanguínea , Uniones Estrechas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Animales Modificados Genéticamente , Antígenos Transformadores de Poliomavirus/genética , Antígenos Transformadores de Poliomavirus/metabolismo , Apoptosis , Barrera Hematonerviosa/citología , Western Blotting , Encéfalo/irrigación sanguínea , Línea Celular , Resistencia a Medicamentos , Impedancia Eléctrica , Femenino , Inmunohistoquímica , Inulina/metabolismo , Pulmón/irrigación sanguínea , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Pericitos/efectos de los fármacos , Puromicina/farmacología , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia Arriba
9.
Cell Struct Funct ; 32(2): 139-47, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18057801

RESUMEN

In autoimmune disorders of the peripheral nervous system (PNS) such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy, breakdown of the blood-nerve barrier (BNB) has been considered as a key step in the disease process. Hence, it is important to know the cellular property of peripheral nerve microvascular endothelial cells (PnMECs) constituting the bulk of BNB. Although many in vitro models of the blood-brain barrier (BBB) have been established, very few in vitro BNB models have been reported so far. We isolated PnMECs from transgenic rats harboring the temperature-sensitive SV40 large T-antigen gene (tsA58 rat) and investigated the properties of these "barrier-forming cells". Isolated PnMECs (TR-BNBs) showed high transendothelial electrical resistance and expressed tight junction components and various types of influx as well as efflux transporters that have been reported to function at BBB. Furthermore, we confirmed the in vivo expression of various BBB-forming endothelial cell markers in the endoneurium of a rat sciatic nerve. These results suggest that PnMECs constituting the bulk of BNB have a highly specialized characteristic resembling the endothelial cells forming BBB.


Asunto(s)
Barrera Hematonerviosa/citología , Células Endoteliales/fisiología , Animales , Animales Modificados Genéticamente , Antígenos Transformadores de Poliomavirus/genética , Biomarcadores , Barrera Hematoencefálica/metabolismo , Barrera Hematonerviosa/metabolismo , Cerebro/metabolismo , Impedancia Eléctrica , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Nervio Ciático/citología , Uniones Estrechas/metabolismo , Venas Umbilicales/citología
10.
Nat Methods ; 4(2): 131-3, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17220894

RESUMEN

Although the laboratory rat (Rattus norvegicus) is an indispensable experimental animal for biomedical research and drug development, the lack of embryonic stem cell lines hampers gene-knockout studies. Here we report the successful generation of insertional mutant rats using the Sleeping Beauty (SB) transposon system. This would benefit a variety of biomedical research fields for which the rat model is better suited than the mouse model.


Asunto(s)
Animales Modificados Genéticamente , Elementos Transponibles de ADN , Mutagénesis Insercional , Ratas/genética , Adenosina Trifosfatasas/genética , Animales , Femenino , Genes Letales , Proteínas Fluorescentes Verdes/genética , Proteínas HSP70 de Choque Térmico/genética , Homocigoto , Masculino , Ratones , Modelos Animales , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Transposasas/genética
11.
Transgenic Res ; 16(1): 115-20, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17103241

RESUMEN

Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.


Asunto(s)
Proteínas Fluorescentes Verdes/genética , Conejos/genética , Animales , Animales Modificados Genéticamente , Cruzamiento , Femenino , Fluorescencia , Expresión Génica , Ingeniería Genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Hibridación Fluorescente in Situ , Masculino , Conejos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Distribución Tisular
12.
Cell Tissue Res ; 326(3): 749-58, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16773315

RESUMEN

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33 degrees C. A temperature shift to 37 degrees C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro.


Asunto(s)
Células Endoteliales/citología , Conducto Torácico/citología , Vena Cava Inferior/citología , Animales , Animales Modificados Genéticamente , Antígenos Virales de Tumores/genética , Biomarcadores/análisis , Técnicas de Cultivo de Célula , Línea Celular Transformada , Proliferación Celular , Transformación Celular Viral , Células Clonales , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Endotelio Linfático/citología , Endotelio Vascular/citología , Colorantes Fluorescentes/metabolismo , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Ratas , Virus 40 de los Simios/fisiología , Temperatura
13.
Proc Natl Acad Sci U S A ; 103(10): 3716-21, 2006 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-16537451

RESUMEN

Three mammalian Period (Per) genes, termed Per1, Per2, and Per3, have been identified as structural homologues of the Drosophila circadian clock gene, period (per). The three Per genes are rhythmically expressed in the suprachiasmatic nucleus (SCN), the central circadian pacemaker in mammals. The phases of peak mRNA levels for the three Per genes in the SCN are slightly different. Light sequentially induces the transcripts of Per1 and Per2 but not of Per3 in mice. These data and others suggest that each Per gene has a different but partially redundant function in mammals. To elucidate the function of Per1 in the circadian system in vivo, we generated two transgenic rat lines in which the mouse Per1 (mPer1) transcript was constitutively expressed under the control of either the human elongation factor-1alpha (EF-1alpha) or the rat neuron-specific enolase (NSE) promoter. The transgenic rats exhibited an approximately 0.6-1.0-h longer circadian period than their wild-type siblings in both activity and body temperature rhythms. Entrainment in response to light cycles was dramatically impaired in the transgenic rats. Molecular analysis revealed that the amplitudes of oscillation in the rat Per1 (rPer1) and rat Per2 (rPer2) mRNAs were significantly attenuated in the SCN and eyes of the transgenic rats. These results indicate that either the level of Per1, which is raised by overexpression, or its rhythmic expression, which is damped or abolished in over expressing animals, is critical for normal entrainment of behavior and molecular oscillation of other clock genes.


Asunto(s)
Ritmo Circadiano/genética , Proteínas Nucleares/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Conducta Animal/fisiología , Proteínas de Ciclo Celular , Ritmo Circadiano/fisiología , Ojo/metabolismo , Femenino , Expresión Génica , Masculino , Ratones , Proteínas Nucleares/fisiología , Proteínas Circadianas Period , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/fisiología
14.
Exp Anim ; 53(4): 395-7, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15297716

RESUMEN

We investigated whether refined follicle stimulating hormone (FSH) with only a little contaminating LH can promote the responsiveness of rabbits to multiple-ovulation treatment. One group of female rabbits was stimulated with refined porcine FSH (pFSH), an FSH source with low LH activity, and another group was treated with pFSH. The mean number of eggs recovered from donors stimulated with refined pFSH (27 +/- 3) was significantly greater (P<0.05) than that with pFSH (20 +/- 2). Furthermore, the mean number of remaining follicles of donors stimulated with refined pFSH (19 +/- 4) was significantly greater (P<0.05) than that with pFSH (12 +/- 1). To decrease the number of remaining follicles in donors treated with refined pFSH, the dose of human chorionic gonadotropin (hCG) was increased from 75 to 150. However, there were no differences in the numbers of eggs and remaining follicles. The results of the present study suggest that refined pFSH with little contaminating LH promotes the responsiveness of rabbits to multiple-ovulation treatment compared with pFSH.


Asunto(s)
Hormona Folículo Estimulante/farmacología , Ovulación/efectos de los fármacos , Superovulación/fisiología , Animales , Femenino , Hormona Folículo Estimulante/aislamiento & purificación , Embarazo , Conejos
15.
J Reprod Dev ; 50(6): 711-5, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15647624

RESUMEN

To improve the efficiency of transgenesis, we investigated the effects of a radical scavenger during microinjection on the development to blastocysts or pups of mouse pronuclear embryos, microinjected with the enhanced green fluorescent protein (EGFP) transgene. When embryos were microinjected in medium containing 0-1,000 units/ml catalase, the developmental rate to blastocysts was significantly higher (P<0.01) in 100-units/ml catalase (81%) than those in 0 and 1,000 units/ml (56 and 65%). To investigate the ontogenetic ability of DNA-injected embryos, EGFP-injected embryos manipulated under 0 or 100 units/ml catalase were transferred separately to recipient mice. The proportion of fetuses derived from EGFP-injected embryos manipulated under 100 units/ml catalase (29%) was significantly higher (P<0.05) than that manipulated under 0 units/ml catalase (19%). Furthermore, the numbers of transgenic pups were 17 in 100 units/ml catalase and 14 in 0 units/ml catalase. The results of the present study indicate that scavenging reactive oxygen species during in vitro micromanipulation is beneficial for the development of DNA-injected embryos.


Asunto(s)
Catalasa/metabolismo , ADN/metabolismo , Técnicas de Cultivo de Embriones/métodos , Técnicas de Transferencia de Gen , Animales , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Técnicas Genéticas , Genotipo , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno , Espermatozoides/metabolismo , Transgenes
16.
Biochem Biophys Res Commun ; 311(4): 920-8, 2003 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-14623269

RESUMEN

Suicide gene expression in specific tissue of transgenic animals has been used for cell-specific ablation. To examine the influence of hepatocyte removal, we produced the herpes simplex virus thymidine kinase (HSVtk) transgenic rat, whose gene was regulated by an albumin enhancer promoter. The liver presence of HSVtk was demonstrated in one line of the transgenic rats. We injected ganciclovir (GCV, 50mg/kg) into the rat on alternate days. After 28 days of GCV administration, liver tissues, and blood of the rats were collected. The histological investigation revealed infiltration of T cells, macrophages, granulocytes/neutrophils, and hepatocyte cell death. The biochemistry analysis demonstrated elevated levels of AST, ALT, and total bilirubin in transgenic rat. In conclusion, the transgenic rat with expressed albumin-specific HSVtk developed experimental hepatitis with administration of GCV, and will be a useful model to facilitate the evaluation of drug effects for clinical control of liver disease.


Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Genes Transgénicos Suicidas/genética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Apoptosis/genética , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ganciclovir/toxicidad , Regulación de la Expresión Génica , Modelos Animales , Ratas/genética , Simplexvirus/enzimología , Proteínas Virales/genética , Proteínas Virales/metabolismo
17.
Biochem Biophys Res Commun ; 305(4): 904-8, 2003 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-12767916

RESUMEN

Animals transgenic (Tg) for reporter genes would be useful to following a given cell lineage during differentiation and regeneration processes. Here, we established a beta-galactosidase (lacZ) Tg rat to use as a tool for regenerative research. Strong lacZ expression was observed in the skeletal muscles, myocardium, pancreas, and skin obtained from these lacZ-Tg rats, and moderate lacZ expression was observed in the liver, spleen, kidney, and cartilage. In contrast, brain, vessels, lung, adrenal gland, small intestine, blood leukocytes, bone marrow (BM) cells, and peripheral blood cells showed no lacZ expression. To test whether this lacZ-Tg rat could be used for regenerative research in myocardium, we induced myocardial injury after a lacZ-Tg BM transplant (BMT) into wild-type rats. The results show that lacZ-positive cardiomyocytes were found in the peri-infarct and uninjured myocardium in the BMT recipient rats. These findings suggest that lacZ-Tg rats are useful tool for regenerative research in the myocardium.


Asunto(s)
Genes Reporteros , Modelos Animales , Miocitos Cardíacos/fisiología , Ratas/genética , Regeneración , beta-Galactosidasa/genética , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea , Cardiomiopatías/etiología , Diferenciación Celular , Masculino , Miocitos Cardíacos/citología , Ratas/anatomía & histología , Ratas/metabolismo , Ratas Wistar , Distribución Tisular , beta-Galactosidasa/análisis
18.
Cell Struct Funct ; 28(1): 87-95, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12655154

RESUMEN

Transgenic mice and rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene are useful for establishing cell lines from tissues. We succeeded in establishing a conditionally immortalized testicular Sertoli cell line, RT3-3, from adult transgenic rats harboring the oncogene. The cells grew at permissive (33 degrees C) and intermediate (37 degrees C) temperatures but not at nonpermissive temperature (39 degrees C). Large T-antigen was expressed at 33 and 37 degrees C, whereas the expression level was gradually decreased at 39 degrees C, suggesting that the temperature-sensitive growth characteristics arise as a result of the function of tsSV40 large T-antigen. The cells showed biochemical features associate with normal Sertoli cells including expressions of mRNAs of sulfated glycoprotein-2 (SGP-2), transferrin (TF) and steel factor. Quantitative polymerase chain reaction revealed that nonpermissive temperature induced increase in the level of SGP-2. Moreover, levels of SGP-2 and/or TF were significantly elevated in the cells treatment with sodium butyrate and retinoic acid, inducers of cellular differentiation. To our knowledge, this is the first report of the establishment of a testicular Sertoli cell line from the transgenic rats. Thus, the conditionally immortalized cell line RTS3-3 with unique characteristics may serve as good experimental in vitro models for basic and applied biology of testicular Sertoli cells.


Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Temperatura Corporal/fisiología , División Celular/genética , Línea Celular Transformada/metabolismo , Oncogenes/genética , Células de Sertoli/metabolismo , Animales , Animales Modificados Genéticamente , Ácido Butírico/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , División Celular/efectos de los fármacos , Línea Celular Transformada/citología , Línea Celular Transformada/efectos de los fármacos , Clusterina , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Glicoproteínas/genética , Masculino , Modelos Biológicos , Chaperonas Moleculares/genética , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Células de Sertoli/citología , Células de Sertoli/efectos de los fármacos , Factor de Células Madre/genética , Transferrina/genética , Tretinoina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
19.
Gene ; 305(1): 71-8, 2003 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-12594043

RESUMEN

Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The -6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.


Asunto(s)
Región de Flanqueo 5'/genética , Regulación de la Expresión Génica/genética , Lactalbúmina/genética , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Levadura/genética , ADN/química , ADN/genética , ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Dosificación de Gen , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ratas , Análisis de Secuencia de ADN
20.
Cell Struct Funct ; 27(2): 71-9, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12207048

RESUMEN

Conditionally immortalized gastric epithelial cell lines were established from transgenic rats harboring temperature-sensitive simian virus 40 (tsSV40) large T-antigen gene. Gastric mucosal cells and epithelial tissues isolated from the stomach of the transgenic rats were cultured at permissive temperature (33 degrees C), and proliferative cells were cloned by colony formation. Six cell lines (designated as RGE1-01, RGE1-02, RGE1-03, RGE1-21, RGE1-22 and RGE2-01) showing epithelial-like morphology have been established. All cells grew at 33 degrees C, but did not at nonpermissive temperature (39 degrees C). High expression level of large T-antigen in the nuclei was observed at 33 degrees C, whereas the expression level was gradually decreased in a time-dependent manner at 39 degrees C. These results suggest that the temperature-sensitive growth characteristics arise as a result of a function of the tsSV40 large T-antigen. None of the cell lines were transformed as judged by anchorage-independent growth assay. Immunocytochemical findings indicated that all cells expressed epithelial cell markers including cytoskeletal (cytokeratin and actin), basement membrane (laminin and collagen type IV) and junctional complex (ZO-1 and desmoplakin I+II) proteins at 33 degrees C. All cells expressed mRNA of cathepsin E, a pit cell marker. Moreover, transepithelial resistance was observed between apical and basolateral sides in the cells. RGE1-22 cells produced prostaglandin E(2). Levels of mRNA for cathepsin E, transepithelial resistance and prostaglandin E(2) were influenced by the nonpermissive temperature. Thus, these conditionally immortalized gastric cell lines which preserve some epithelial cell characteristics will provide a useful in vitro model of gastric epithelium.


Asunto(s)
Antígenos Virales de Tumores/genética , Línea Celular Transformada , Células Epiteliales/citología , Virus 40 de los Simios/genética , Animales , Animales Modificados Genéticamente , Biomarcadores , Catepsina E/genética , División Celular , Dinoprostona/análisis , Impedancia Eléctrica , Mucosa Gástrica/citología , Masculino , ARN Mensajero/análisis , Ratas , Ratas Wistar , Temperatura
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...