RESUMEN
IMPORTANCE: Cryptococcosis studies often utilize the common C57BL/6J mouse model. Unfortunately, infection in these mice fails to replicate the basic course of human disease, particularly hampering immunological studies. This work demonstrates that SJL/J mice can recapitulate human infection better than other mouse strains. The immunological response to Cryptococcus infection in SJL/J mice was markedly different from C57BL/6J and much more productive in combating this infection. Characterization of infected mice demonstrated strain-specific genetic linkage and differential regulation of multiple important immune-relevant genes in response to Cryptococcus infection. While our results validate many of the previously identified immunological features of cryptococcosis, we also demonstrate limitations from previous mouse models as they may be less translatable to human disease. We concluded that SJL/J mice more faithfully recapitulate human cryptococcosis serving as an exciting new animal model for immunological and genetic studies.
Asunto(s)
Criptococosis , Cryptococcus neoformans , Humanos , Ratones , Animales , Cryptococcus neoformans/genética , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
Neoplastic tumors of the ocular lens of vertebrates do not naturally occur. Transgenic mice carrying a hybrid gene comprising the murine alpha A-crystallin promoter (-366 to +46) fused to the coding sequence of the SV40 T antigens developed lens tumors, which obliterated the eye cavity and even invaded neighboring tissue, thus establishing that the lens is not refractive to oncogenesis. Large-T antigen was detected early in lens development; it elicited morphological changes and specifically interfered with differentiation of lens fiber cells. Both alpha- and beta-crystallins persisted in many of the lens tumor cells, while gamma-crystallin was selectively reduced. Accessibility, characteristic morphology, and defined protein markers make this transparent epithelial eye tissue a potentially useful system for testing tumorigenicity of oncogenes and for studying malignant transformation from its inception until death of the animal.
Asunto(s)
Neoplasias del Ojo/patología , Enfermedades del Cristalino/patología , Ratones/genética , Animales , Antígenos Transformadores de Poliomavirus , Antígenos Virales de Tumores/análisis , Transformación Celular Neoplásica , Transformación Celular Viral , Quimera , Cristalinas/análisis , Femenino , Cristalino/crecimiento & desarrollo , Proteínas Oncogénicas Virales/análisis , Fenotipo , Embarazo , Virus 40 de los Simios , Transcripción GenéticaRESUMEN
Phenotype variability and incomplete penetrance are frequently observed in human monogenic diseases such as osteogenesis imperfecta. Here an inbred strain of transgenic mice expressing an internally deleted gene for the pro alpha 1(I) chain of type I procollagen (COL1A1) was bred to wild type mice of the same strain so that the inheritance of a fracture phenotype could be examined in a homogeneous genetic background. To minimize the effects of environmental factors, the phenotype was evaluated in embryos that were removed from impregnated females 1 d before term. Examination of stained skeletons from 51 transgenic embryos from 11 separate litters demonstrated that approximately 22% had a severe phenotype with extensive fractures of both long bones and ribs, approximately 51% had a mild phenotype with fractures of ribs only, and approximately 27% had no fractures. The ratio of steady-state levels of the mRNA from the transgene to the level of mRNA from the endogenous gene was the same in all transgenic embryos. The results demonstrated that the phenotypic variability and incomplete penetrance were not explained by variations in genetic background or levels in gene expression. Instead, they suggested that phenotypic variation is an inherent feature of expression of a mutated collagen gene.
Asunto(s)
Colágeno/genética , Fracturas Óseas/genética , Animales , Secuencia de Bases , Femenino , Fracturas Óseas/etiología , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Fenotipo , ARN Mensajero/análisisRESUMEN
A line of transgenic mice was prepared that expressed moderate levels of an internally deleted human gene for the pro alpha 1(I) chain of type I procollagen. The gene construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta by causing biosynthesis of shortened pro alpha 1(I) chains. 89 transgenic mice from the line were examined. About 6% had a lethal phenotype with extensive fractures at birth, and 33% had fractures but were viable. The remaining 61% of the transgenic mice had no apparent fractures as assessed by x ray examination on the day of birth. Brother-sister matings produced eight litters in which approximately 40% of the mice had the lethal phenotype, an observation indicating that expression of the exogenous gene was more lethal in putative homozygous mice from the line. Examination of femurs from the transgenic mice indicated that the bones were significantly shorter in length and had a decrease in wet weight, mineral content, and collagen content. However, there was no statistically significant change in the mineral to collagen ratio. Biomechanical measurements on femurs from the mice at 6 wk indicated a decrease in force and energy to failure. There was also a decrease in strain to failure and an increase in Young's modulus of elasticity, observations indicating increased brittleness of bone matrix. The results suggested that the transgenic mice may be an appropriate model for testing potential therapies for osteogenesis imperfecta. They may also be a useful model for studying osteoporosis.
Asunto(s)
Densidad Ósea/genética , Huesos/química , Colágeno/análisis , Fracturas Espontáneas/genética , Eliminación de Gen , Procolágeno/genética , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , FenotipoRESUMEN
Studies were carried out on a line of transgenic mice that expressed an internally deleted COL2A1 gene and developed a phenotype resembling human chondrodysplasias (Vandenberg et al. 1991. Proc. Natl. Acad. Sci. USA. 88:7640-7644. Marked differences in phenotype were observed with propagation of the mutated gene in an inbred strain of mice in that approximately 15% of the transgenic mice had a cleft palate and a lethal phenotype, whereas the remaining mice were difficult to distinguish from normal littermates. 1-d- and 3-mo-old transgenic mice that were viable showed microscopic signs of chondrodysplasia with reduced amounts of collagen fibrils in the cartilage matrix, dilatation of the rough surfaced endoplasmic reticulum in the chondrocytes, and decrease of optical path difference in polarized light microscopy. The transgenic mice also showed signs of disturbed growth as evidenced by lower body weight, lower length and weight of the femur, decreased bone collagen, decreased bone mineral, and decreased resistance of bone to breakage. Comparisons of mice ranging in age from 1 d to 15 mo demonstrated that there was decreasing evidence of a chondrodysplasia as the mice grew older. Instead, the most striking feature in the 15-mo-old mice were degenerative changes of articular cartilage similar to osteoarthritis.
Asunto(s)
Cartílago/ultraestructura , Eliminación de Gen , Procolágeno/genética , Envejecimiento/fisiología , Animales , Secuencia de Bases , Peso Corporal , Desarrollo Óseo , Huesos/metabolismo , Cartílago/crecimiento & desarrollo , Fisura del Paladar/genética , Colágeno/biosíntesis , Colágeno/metabolismo , Cósmidos , Exones , Matriz Extracelular/ultraestructura , Femenino , Genes Letales , Placa de Crecimiento/ultraestructura , Humanos , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Microscopía Electrónica , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Linaje , Reacción en Cadena de la Polimerasa , Valores de Referencia , Mapeo Restrictivo , Factores SexualesRESUMEN
Work by a large number of investigators over the last decade has established that over 90% of patients with osteogenesis imperfecta have mutations in one of the two genes for type I procollagen, that most unrelated probands have different mutations in the genes, and that the mutations found in most of the serious variants of the disease cause synthesis of abnormal pro alpha chains of the protein. The results have demonstrated that synthesis of structurally abnormal but partially functional pro alpha chains can interfere with folding of the central region of the protein into a triple-helical conformation, prevent processing of the N-terminal propeptides of procollagen, or produce subtle alterations in conformation that interfere with the self-assembly of the protein into collagen fibrils. One of the unsolved mysteries about the disease is why some mutations produce severe phenotypes, whereas very similar mutations produce mild phenotypes. Recent studies in transgenic mice suggest that nongenetic factors, such as stochastic events during development, may determine the severity of the disease phenotype produced by a specific mutation. Also, recent results raised the possibility that strategies of antisense gene therapy may be effective in treating the disease some time in the future. Specific inhibition of expression of a mutated collagen gene has been obtained with antisense oligonucleotides in cell culture experiments. However, there is no means of selective delivery of antisense oligonucleotides to the appropriate tissues.
Asunto(s)
Colágeno/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , Osteogénesis Imperfecta/genética , Procolágeno/genética , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Mutación , Oligonucleótidos Antisentido/farmacología , Osteogénesis Imperfecta/tratamiento farmacológico , Osteogénesis Imperfecta/etiología , Fenotipo , Procolágeno/químicaRESUMEN
A line of transgenic mice have been investigated that expressed moderate levels of an internally deleted human gene for the pro alpha (I) chain of type I procollagen. These mice expressed the gene at approximately 50% that of the endogenous gene. The gene construct was modeled after a sporadic in-frame deletion of the human gene that produced a lethal variant of osteogenesis imperfecta by causing biosynthesis of shortened pro alpha (I) chains. Periera et al. (1993) reported extensive fracturing in these mice with femurs that were shorter in length and bone that had decreased ash weight, mineral, and collagen content. These workers demonstrated an increased brittleness in bone using biomechanical measurements. The functional consequences of these mutant genes were examined in both transgenic and in normal littermate mice to determine if a valid model at the ultrastructural and analytical level had been produced for OI. X-ray microanalysis of bone mineral demonstrated a significantly lower calcium-to-phosphorus (Ca/P) molar ratio in transgenic mouse bone than in normal littermates; this was a feature of human OI bone. Fourier transform infrared spectroscopy confirmed that the mineral present was apatitic in nature despite the lower Ca/P molar ratio. Alizarin red skeletal staining showed the presence of multiple fracture calluses on the ribs and on the long bones of some of the transgenic mice, this was not seen on normal littermates. No light microscopic differences were observed between normal and transgenic mice; however, many ultrastructural correlates with human OI were observed in the transmission electron microscope. Anomalous fibrils associated with type I collagen, and an amorphous calcified material was observed lining the cartilage, extending beyond the lamina limitans in young transgenic mice.
Asunto(s)
Fémur/ultraestructura , Regulación de la Expresión Génica/genética , Placa de Crecimiento/ultraestructura , Procolágeno/genética , Envejecimiento/metabolismo , Animales , Densidad Ósea/fisiología , Calcio/metabolismo , Modelos Animales de Enfermedad , Microanálisis por Sonda Electrónica , Fémur/metabolismo , Placa de Crecimiento/metabolismo , Ratones , Ratones Transgénicos , Microscopía Electrónica , Modelos Genéticos , Mutación/genética , Osteoblastos/citología , Osteoblastos/ultraestructura , Osteogénesis Imperfecta/genética , Fósforo/metabolismo , Regiones Promotoras Genéticas , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A single-step coculture procedure has been developed that can generate chimeric mice with high efficiency and reproducibility. The procedure involves culture of embryonic stem (ES) cells with 8- to 16-cell embryos in microwells to provide conditions for effective cell-cell-embryo contact. A suspension of ES cells is layered over the microwells, followed by transfer of an embryo without zona pellucida into each microwell. Following overnight culture, the blastocysts are transferred into pseudopregnant recipients. The method has several advantages due to its simplicity and reproducibility: (i) Over 90% of ES-cell contribution in new-borns can be obtained frequently and most of the chimeras display germ-line transmission. (ii) The procedure does not require specialized skills or expensive instruments. (iii) All the steps of embryo manipulation can be completed in a relatively short period of time; therefore, a large number of embryos can be manipulated simultaneously. The method was tested with three independent ES cell lines and two different strains of mice with similar results. The technique may be an alternative to microinjection of DNA into zygotes to prepare transgenic lines of small and large mammalian species.
Asunto(s)
Blastocisto/fisiología , Quimera/genética , Técnicas de Cocultivo/métodos , Células Madre/citología , Animales , Blastocisto/citología , Células Cultivadas , Transferencia de Embrión , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Four lines of transgenic mice containing the HIV LTR linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT) were constructed. In each line, a characteristic tissue pattern of CAT expression was observed with detectable levels present in the eye, heart, spleen, thymus, and tail. Low levels of CAT were present in circulating lymphocytes, but CAT activity in these cells could be augmented following treatment with the mitogen phytohemagglutinin (PHA). Likewise, CAT expression was present at only low levels in circulating monocytes, but higher levels of CAT were observed in macrophages grown in the presence of various cytokines (CSF-1, GM-CSF, IL-1 alpha, IL-4, and IL-2). Furthermore, Langerhans cells recovered from skin showed higher levels of CAT activity than those observed in other cells of monocyte-macrophage lineage. These results indicate that LTR-CAT expression in cells of monocyte-macrophage lineage may increase in proportion to the degree of differentiation of these cells. These animals may be useful in the study of cell-specific determinants of LTR-directed gene activity and may serve to identify exogenous cofactors that promote the progression of HIV-related disease in vivo.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/biosíntesis , VIH/genética , Células de Langerhans/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Cloranfenicol O-Acetiltransferasa/genética , Productos del Gen tat , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Linfocitos T/inmunología , Factores de Transcripción/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The abundant soluble proteins of the eye lens, the crystallins, are encoded by several gene families which are developmentally regulated in the embryonic lens. We have studied the expression of the murine alpha A-crystallin gene. Transfection experiments using the pSVO-CAT vector and explanted lens epithelia from embryonic chickens demonstrated proximal (-88 to -60) and distal (-111 to -85) regulatory sequences which interact when the alpha A-crystallin promoter is activated in the lens cells. Transgenic mouse experiments showed that the sequence between positions -366 to +46 of the alpha A-crystallin promoter can drive foreign genes selectively in the lens. A fusion gene consisting of this alpha A-crystallin promoter sequence and the T-antigen gene of SV40 produced a lens tumor in transgenic mice. Thus, crystallin promoters provide a useful model for tissue-specific gene expression and permit targeting the expression of foreign genes to a highly differentiated tissue during development.
Asunto(s)
Regulación de la Expresión Génica , Cristalino/fisiología , Animales , RatonesRESUMEN
Mos is a germ cell-specific serine/threonine protein kinase that plays an important role during meiotic divisions of oocytes. Upon expression in somatic cells, Mos causes cell cycle perturbations leading to neoplastic transformation. Mos activates the MAP kinase pathway in both oocytes and transformed somatic cells. To determine the mechanism of cell cycle perturbation in mos-transformed cells, we examined the status of some key regulators of G1 phase. We provide evidence that Mos causes an elevation in the level of cyclin D1 in NIH/3T3 cells. As expected from the increased cyclin D1 level, mos transformation of NIH/3T3 cells caused an increase in the protein kinase activities of cyclin D1-Cdk4 and cyclin E-Cdk2 and induced hyperphosphorylation of the retinoblastoma protein. Of importance, the level of cyclin D1 was also elevated in eye lens of the c-mos-transgenic mice compared to normal mice. Our results indicate that the mechanism of cellular transformation by Mos involves an elevation in the level of cyclin D1 in somatic cells.
Asunto(s)
Quinasas CDC2-CDC28 , Transformación Celular Neoplásica , Ciclina D1/biosíntesis , Genes mos , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Células 3T3 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Cristalino/metabolismo , Meiosis , Ratones , Ratones Transgénicos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mos/biosíntesis , Transducción de SeñalAsunto(s)
Clonación Molecular , ADN/genética , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos , Integración Viral/genética , Animales , Secuencia de Bases , ADN Complementario/genética , ADN Recombinante/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , TransgenesAsunto(s)
Trasplante de Células/métodos , Células Germinativas/citología , Animales , Quimera , HumanosRESUMEN
A recombinant plasmid containing the Drosophila melanogaster P element transposon was microinjected into mouse zygotes. Dot-blot analysis indicated that one of the newborns contained a single copy of the microinjected DNA per haploid mouse genome equivalent; two other newborns had integrated multiple copies of the P element construct. Southern mapping revealed that the entire plasmid, including both pBR322 sequences and P element sequences, had integrated in each of the three animals. In the two mice carrying multiple copies of the microinjected DNA, the copies appear to be linked in a tandem head-to-tail array. Therefore, in each of the three newborns integration of P element sequences has occurred by a mechanism which is distinct from that observed when the same plasmid is injected into Drosophila embryos. Analysis of DNA from the offspring of one of the transgenic mice showed no indication of transposition of P element sequences.
Asunto(s)
Drosophila melanogaster/genética , Ratones/genética , Transformación Genética , Animales , ADN/genética , Femenino , Masculino , Microinyecciones , PlásmidosRESUMEN
Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes.
Asunto(s)
Enfermedades Óseas/genética , Quimera , Colágeno/genética , ADN sin Sentido , Genes Letales , Genes Sintéticos , Procolágeno/genética , Animales , Secuencia de Bases , Western Blotting , Colágeno/biosíntesis , Colágeno/aislamiento & purificación , Cartilla de ADN , Femenino , Humanos , Intrones , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
Previous reports have provided inconsistent data as to the cis-regulatory elements that are essential for correct expression of the gene for the pro alpha 1 (I) chain of type I procollagen (COL1A1) in the many tissues in which the protein is synthesized. Here, two internally deleted minigene versions of the human COL1A1 gene were used to prepare transgenic mice. The constructs made it possible to test regulatory sequences in the normal context of the gene. Also, in contrast to the reporter genes used in previous experiments, the constructs made it possible to assay quantitatively expression of the exogenous genes relative to expression of the endogenous COL1A1 gene, both as mRNA and as protein. The average level of expression of the minigenes varied among three transgenic lines, but the ratio of expression of the minigenes to expression of the endogenous gene was the same in all transgenic mice of a given line. Within the same line, the ratio of expression was essentially the same in nine or more tissues in which expression of the endogenous gene varied widely. Also, the ratio of expression within a given line was the same in 15-day-old embryos and in mice ranging in age from 4 days to 4 months. In addition, the ratio remained constant during repair of a surgical wound. The results demonstrated, therefore, that the minigene constructs with about 2.3 kb of the promoter region and about 2 kb of the 3'-flanking region contained all of the sequences necessary for correct expression of the genes in a tissue-specific and development-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Genes/genética , Genoma Humano , Ratones Transgénicos , Procolágeno/genética , Animales , Secuencia de Bases , Amplificación de Genes , Regulación de la Expresión Génica , Humanos , Intrones/genética , Ratones , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Procolágeno/análisis , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Secuencias Reguladoras de Ácidos Nucleicos/genética , Eliminación de Secuencia , Cicatrización de Heridas/fisiologíaRESUMEN
Eight transgenic mice were generated in which the promoter of the mouse alpha 2(I) collagen gene (nucleotides -2000 to +54), linked to the bacterial gene for chloramphenicol acetyltransferase (CAT), is stably integrated in the germ line. These strains contain from 1 to 20 copies of the alpha 2(I) collagen-CAT chimeric gene per haploid genome. In seven of the eight strains, the CAT gene is expressed, although the levels of CAT enzyme activity vary considerably from one strain to the other. In six of these strains, the expression of the CAT gene follows the expected tissue distribution pattern of expression of the alpha 2(I) collagen gene. In these six strains, the level of CAT activity is much higher in extracts of tail, a tissue that is very rich in tendons, than in any other tissue that was tested. This distribution parallels the much higher levels of alpha 2(I) collagen RNA that are found in the tail as compared to other tissues. Expression of the chimeric gene is detected in the embryo after 8.5 days of gestation, at approximately the same time that the endogenous type I genes become active. We conclude that the alpha 2(I) collagen promoter sequences present in the recombinant plasmid used for our experiments contain sufficient information to ensure stage- and tissue-specific activity of this promoter.
Asunto(s)
Colágeno/genética , Regulación de la Expresión Génica , Genes Reguladores , Regiones Promotoras Genéticas , Acetiltransferasas/biosíntesis , Acetiltransferasas/genética , Animales , Cloranfenicol O-Acetiltransferasa , Colágeno/biosíntesis , ADN Recombinante , Transferencia de Embrión , Femenino , Ingeniería Genética , Masculino , Ratones , Especificidad de Órganos , Plásmidos , EmbarazoRESUMEN
The authors describe an ES cell technique that resolves the problems of time, expense, and inconsistency often encountered in the production of transgenic mice via DNA microinjection.