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At least 343 records · Page 19Linked to original sources
PubMed2002

Doxycycline-induced expression of sense and inverted-repeat constructs modulates phosphogluconate mutase (Pgm) gene expression in adult Drosophila melanogaster.

BACKGROUND: A tetracycline-regulated (conditional) system for RNA interference (RNAi) would have many practical applications. Such a strategy was developed using RNAi of the gene for phosphogluconate mutase (Pgm). Pgm is a candidate lifespan regulator: PgmS allele frequency is increased by selection for increased lifespan, whereas PgmM and PgmF allele frequencies are decreased. RESULTS: The Pgm alleles were cloned and sequenced and were found to differ by amino-acid substitutions consistent with the relative electrophoretic mobilities of the proteins. The 'tet-on' doxycycline-regulated promoter system was used to overexpress PgmS in a wild-type (PgmM) background. Enzyme activity increases of two- to five-fold were observed in five independent transgenic lines. Tet-on was also used to drive expression of an inverted-repeat fragment of Pgm coding region. The inverted-repeat transcript was expected to form a dsRNA hairpin, induce RNAi, and thereby reduce endogenous Pgm gene expression at the RNA level. Endogenous Pgm RNA levels in adult flies were found to be reduced or eliminated by doxycycline treatment in five independent inverted-repeat transgenic lines. Our results show that doxycycline-regulated expression of inverted-repeat constructs can cause a conditional reduction in specific gene expression. The effect of sense and inverted-repeat construct expression on lifespan was assayed in multiple transgenic lines. Under the conditions tested, altered Pgm gene expression had no detectable effect on adult Drosophila lifespan. CONCLUSIONS: A system for conditional RNAi in Drosophila adults shows promise for assay of gene functions during aging. Our results indicate that Pgm does not have a simple strong effect on longevity.

Aging↗
PubMed2003

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress.

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northern blot, revealed variable levels of transgene expression. Antibody probing of Western blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significant increase in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide the first biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.

Arabidopsis↗
PubMed2003

Stable transgene expression and random gene silencing in wheat.

Wheat genes for pathogenesis-related (PR-)proteins, chitinase and beta-1,3-glucanase, under the control of maize ubiquitin promoter-intron were used for transforming the spring wheat 'Bobwhite', using a biolistic approach. Twenty of the 24 primary transgenic lines expressing the PR-protein genes in the T0 generation were silenced in either the T1 or T2 generations. Two apparently genetically identical regenerants arising from a single callus co-bombarded with chitinase and beta-1,3-glucanase transgene combinations, but differing in the expression of the transgenes were selected for further characterization. In one homozygous line, transgene silencing was observed in the T3 plants, while the other line homozygous for the transgene loci stably expressed and inherited the transgenes to at least the T4 generation. Southern blot analyses of genomic DNA from the two lines using the isoschizomeric methylation-sensitive enzymes, MspI and HpaII, revealed a higher degree of methylation of CCGG sequences in the line with the silenced transgene locus. Analysis by reverse transcriptase-polymerase chain reaction, Northern blotting and Western blotting detected stable expression of the transgenes in the line with a lesser extent of methylation, whereas the line with a higher level of CCGG methylation had no transgene expression by the T3 generation. The germination of seeds from the silenced plants in the presence of a cytidine analogue, 5-azacytidine (azaC), did not lead to a reversion of this phenotype.

Journal Article↗
PubMed1990

Tissue-specific expression and methylation of a thyroglobulin-chloramphenicol acetyltransferase fusion gene in transgenic mice.

Fusion genes containing 1600 or 2000 base pairs of the bovine thyroglobulin gene 5' flanking region and the chloramphenicol acetyltransferase (CAT) coding sequence were constructed and used to generate transgenic mice. Altogether, 24 independent transgenic lines were obtained, and the expression of the transgene was assayed by measuring the CAT activity in different tissues. Depending on the transgenic lines, the fusion gene was either silent in all tissues or specifically expressed in the thyroid. The level of expression was found to be highly variable from one line to another and to be regulated by thyrotropin in a manner similar to the natural thyroglobulin gene. The methylation status of the integrated DNA was tested by digestion of DNA extracted from thyroid and other tissues with the isochizomers Msp I and Hpa II. It was found that one of the Hpa II sites was demethylated specifically in the thyroid.

Animals↗
PubMed2006

Overexpression of PKD1 causes polycystic kidney disease.

The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1 gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1 expression have been detected in the kidneys of ADPKD patients. To determine whether Pkd1 gain of function can be a pathogenetic process, a Pkd1 bacterial artificial chromosome (Pkd1-BAC) was modified by homologous recombination to solely target a sustained Pkd1 expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the Pkd1 transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular cysts and renal insufficiency and died of renal failure. This model demonstrates that overexpression of wild-type Pkd1 alone is sufficient to trigger cystogenesis resembling human ADPKD. Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is a critical in vivo downstream effector of Pkd1 molecular pathways. This study not only produced an invaluable and first PKD model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD.

Animals↗
PubMed2004

Mechanism of gene expression of Arabidopsis glutathione S-transferase, AtGST1, and AtGST11 in response to aluminum stress.

The gene expression of two Al-induced Arabidopsis glutathione S-transferase genes, AtGST1 and AtGST11, was analyzed to investigate the mechanism underlying the response to Al stress. An approximately 1-kb DNA fragment of the 5'-upstream region of each gene was fused to a beta-glucuronidase (GUS) reporter gene (pAtGST1::GUS and pAtGST11::GUS) and introduced into Arabidopsis ecotype Landsberg erecta. The constructed transgenic lines showed a time-dependent gene expression to a different degree in the root and/or leaf by Al stress. The pAtGST1::GUS gene was induced after a short Al treatment (maximum expression after a 2-h exposure), while the pAtGST11::GUS gene was induced by a longer Al treatment (approximately 8 h for maximum expression). Since the gene expression was observed in the leaf when only the root was exposed to Al stress, a signaling system between the root and shoot was suggested in Al stress. A GUS staining experiment using an adult transgenic line carrying the pAtGST11::GUS gene supported this suggestion. Furthermore, Al treatment simultaneously with various Ca depleted conditions in root region enhanced the gene expression of the pAtGST11::GUS in the shoot region. This result suggested that the degree of Al toxicity in the root reflects the gene response of pAtGST11::GUS in the shoot via the deduced signaling system. Both transgenic lines also showed an increase of GUS activity after cold stress, heat stress, metal toxicity, and oxidative damages, suggesting a common induction mechanism in response to the tested stresses including Al stress.

Adaptation, Physiological↗
PubMed1990

Cell type-specific expression of JC virus T antigen in primary and established cell lines from transgenic mice.

The highly restricted host range of JC virus (JCV) has made it difficult to study the biology of this common human papovavirus. To increase our understanding of the tissue specificity of this virus, we have examined the expression of the T antigen (T-Ag) in primary and established cell lines from various tissues of transgenic mice containing the JCV early region. In contrast to earlier results from a simian virus 40-containing transgenic mouse, there was no T-Ag expression in mesenchymal fibroblasts derived from two lines of JCV-transgenic mice. Instead, we isolated T-Ag-positive (T-Ag+) cells that had characteristics consistent with a neural crest origin. Furthermore, primary brain cultures contained many T-Ag+ astrocytes, but no expression was detected in macrophages, epithelial cells, neuronal cells nor, surprisingly, in oligodendrocytes. Continued passage of these cultures resulted in vigorously growing glial fibrillary acidic protein-positive, T-Ag+ astrocytes. Thus, the strict tissue specificity of JCV expression was maintained, despite the fact that the viral genome pre-existed in every tissue of these transgenic mice and these constraints on expression were preserved even when cells were explanted in vitro.

Animals↗
PubMed1988

Glomerular epithelial, mesangial, and endothelial cell lines from transgenic mice.

The culture of glomerular cells has represented an important tool in the understanding of individual glomerular cell functions. However, the complexity of the glomerulus has made it difficult to obtain pure cell populations. It has also been difficult to culture glomerular endothelial cells, even as mixed cell populations. At present there are no established glomerular cell lines from any source. We have established permanent cell lines of cloned glomerular epithelial, mesangial, and endothelial cells from a line of mice transgenic for the early region of simian virus 40 (SV40). These mice appear normal at birth but by three to four months of age have sclerosis affecting a variable percentage of their glomeruli. The cells maintain features characteristic of their normal counterparts despite their transformed phenotype. These cell lines could be useful tools in understanding the pathogenesis of glomerulosclerosis in this transgenic mouse model and in studying those features of normal glomerular cell biology which are not altered by a transformed phenotype.

Animals↗
PubMed2006

Adapting rice anther culture to gene transformation and RNA interference.

Anther culture offers a rapid method of generating homozygous lines for breeding program and genetic analysis. To produce homozygous transgenic lines of rice (Oryza sativa L.) in one step, we developed an efficient protocol of anther-callus-based transformation mediated by Agrobacterium after optimizing several factors influencing efficient transformation, including callus induction and Agrobacterium density for co-cultivation. Using this protocol, we obtained 145 independent green transformants from five cultivars of japonica rice by transformation with a binary vector pCXK1301 bearing the rice gene, Xa21 for resistance to bacterial blight, of which 140 were further confirmed by PCR and Southern hybridization analysis, including haploids (32.1%), diploids (62.1%) and mixoploids (7.5%). Fifteen diploids were found to be doubled haploids, which accounted for 10.7% of the total positive lines. Finally, by including 28 from colchicine induced or spontaneous diploidization of haploids later after transformation, a total of 43 doubled haploids (30.7%) of Xa21 transgenic lines were obtained. We also generated two RNAi transgenic haploids of the rice OsMADS2 gene, a putative redundant gene of OsMADS4 based on their sequence similarity, to investigate its possible roles in rice flower development by this method. Flowers from the two OsMADS2 RNAi transgenic haploids displayed obvious homeotic alternations, in which lodicules were transformed into palea/lemma-like tissues, whereas identities of other floral organs were maintained. The phenotypic alternations were proved to result from specific transcriptional suppression of OsMADS2 gene by the introduced RNAi transgene. The results confirmed that OsMADS2 is involved in lodicule development of rice flower and functionally redundant with OsMADS4 gene. Our results demonstrated that rice anther culture could be adapted to gene transformation and RNAi analysis in rice.

Adaptation, Biological↗
PubMed1994

Expression of synthetic cDNA sequences encoding human insulin-like growth factor-1 (IGF-1) in the mammary gland of transgenic rabbits.

We have developed an expression system where foreign proteins are synthesized specifically in the mammary gland of transgenic rabbits and secreted into the milk. Regulatory elements were isolated from the bovine alpha S1-casein-encoding gene and combined with a synthetic DNA coding for human IGF-1 and for [Gln58]IGF-1, an IGF-1 analogue. The resulting hybrid DNA constructs were used to generate transgenic rabbits. Females of seven transgenic lines tested were positive for synthesis of IGF-1. Transmission of the transgene to progeny and IGF-1 production in female offspring was observed in all transgenic lines analysed. As expected, expression of transgene mRNA could only be detected in the mammary gland. Production levels of transgenic protein were as high as 1 g IGF-1 per liter rabbit milk. IGF-1, as well as [Gln58]IGF-1, when secreted into rabbit milk, was correctly processed and biologically active. IGF-1 was purified from the milk of transgenic rabbits to a nearly homogenous active form.

Animals↗
PubMed2004

Overexpression of human erythropoietin (EPO) affects plant morphologies: retarded vegetative growth in tobacco and male sterility in tobacco and Arabidopsis.

Erythropoietin (EPO) is a glycoprotein used for curing human anemia by regulating the differentiation of erythroid progenitors and the production of red blood cells. To examine the expression of recombinant EPO in plants, pPEV-EP21, in which human epo cDNA under the control of the CaMV 35S promoter, was introduced into tobacco and Arabidopsis via Agrobacterium tumefaciens-mediated transformation. The RNA expression level of epo in the transgenic lines was initially estimated by Northern blot analysis. Two transgenic lines, which exhibited a high expression level of epo mRNA determined by Northern analysis, were chosen for Western blot analysis to examine the production of EPO proteins. Those two lines, EP21-12 and EP21-14, revealed detectable bands on the immunoblot. Interestingly, constitutive expression of the human epo gene affected the morphologies in transgenic plants such that vegetative growth of transgenic tobacco was retarded, and male sterility was induced in transgenic tobacco and Arabidopsis.

Agrobacterium tumefaciens↗
PubMed2001

Establishment of gingival epithelial cell lines from transgenic mice harboring temperature sensitive simian virus 40 large T-antigen gene.

We established two gingival epithelial cell lines (GE1 and GE6), originating from transgenic mice harboring the temperature-sensitive simian virus 40 large T-antigen gene. GE1 and GE6 grew at a permissive temperature (33 degrees C) in a pavement arrangement and solely formed multilayers that exhibited morphological features similar to those of the stratified oral epithelium, with neither the use of stromal equivalents nor feeder layers. Both GE cells underwent apoptosis at a non-permissive temperature (39 degrees C). Characteristic keratin peptides, keratin 4 and 13, for mucosal epithelium were obviously expressed in the suprabasal cells, and keratohyalin granules and involucrin were present in the surface flat cells in the multilayered culture. Keratin 10 (one of the markers for higher keratinized gingival epithelium) was rarely found in some uppermost cells, and filaggrin (a component of keratohyalin granules) appeared sparsely in uppermost desquamating cells in the older cultures. These observations indicated that GE1 and GE6 cells exhibited the phenotype characterizing nonkeratinized sulcular epithelium, which possessed the potency undergoing keratinization in such highly stratified cultures as oral gingival epithelium. GE cells increased the expression levels of mRNA of interleukin-1beta and tumor necrosis factor alpha by the stimulation of lipopolysaccharide and extracellular substances of oral streptococci. The GE cell lines thus could serve as an excellent experimental system for further studies on the physiology of gingival epithelium and corresponding diseases, such as periodontal disease, epithelial hyperplasia, and gingival tumors.

Animals↗
PubMed1999

Human growth hormone transgene expression increases the biomechanical structural properties of mouse vertebrae.

STUDY DESIGN: Caudal vertebrae were obtained from male and female mice from two transgenic lines expressing an erythroid-specific human growth hormone transgene construct, and gender-matched, age-matched, non-transgenic control mice. OBJECTIVE: To characterize the effect of human growth hormone transgene expression on the biomechanical structural properties of caudal vertebrae in compression. SUMMARY OF BACKGROUND DATA: An increase in trabecular and cortical bone deposition caused by erythroid-specific human growth hormone transgene expression was demonstrated previously. METHODS: Compression tests were performed on individual caudal vertebrae (Ca4, Ca5, Ca6) obtained from male and female mice from two transgenic lines (TG420 and TG450) and nontransgenic control mice. Two age groups were evaluated: 12 weeks old and 6 months old. The data were used to obtain axial stiffness, maximum load, and energy to failure. RESULTS: Vertebrae from male TG420 transgenic mice produced significantly larger values for maximum load, energy to failure, and axial stiffness at both 12 weeks and 6 months in comparison with their age-matched non-transgenic male controls. Vertebrae from female TG420 transgenic mice produced similar responses at 6 months. Vertebrae from male TG450 transgenic mice showed significant increases in maximum load and energy to failure at 6 months. In general, the biomechanical properties of vertebrae were significantly larger in the 6-month age group than in the 12-week age group, and this increase was significantly greater in the transgenic mice than in the gender-matched control mice during the same time span. This process was also influenced by transgenic genotype and gender. CONCLUSIONS: Erythroid-specific production of human growth hormone in transgenic mice resulted in significant increases in biomechanical properties of their caudal vertebrae in compression. The changes in the biomechanical properties were influenced by genotype, age, and gender.

Aging↗
PubMed1998

Tissue-specific expression of human salivary mucin gene, MUC7, in transgenic mice.

The MUC7 gene encodes the protein core of the low molecular weight human salivary mucin (MG2, mucin glycoprotein 2) and is expressed in a tissue-specific manner in salivary glands. The purpose of this study was to examine MUC7 expression by transgenic mouse technology. A 16 kb DNA fragment, containing the MUC7 gene (10 kb) and 3 kb of the upstream and 3 kb of the downstream sequences, was used to generate transgenic mice. We have identified five transgenic founder mice which were propagated as individual transgenic lines and analysed. Tissues of transgenic offspring from each line were analysed by RT-PCR to determine the sites of the MUC7 expression. The results indicated that only line 3 and line 5 expressed the MUC7 gene in salivary glands. The level of MUC7 expression in selected tissues was then determined by northern blot analyses. The results showed that line 3 mice contained high levels of MUC7 transcripts in the sublingual glands of both males and females and indicated low levels of MUC7 transcripts in the submandibular glands of females. No MUC7 expression was detected in this line by northern blot analysis in any other tissue tested. On the other hand, no expression of MUC7 was detected in any tissues of line 5 mice examined by northern blot analysis. A Southern blot analysis of human and mouse genomic DNA demonstrated multiple copies of the MUC7 transgene in line 3 and a single copy in line 5. Collectively, these results indicate that the regulatory sequences required for the tissue-specific expression of MUC7 are within the MUC7 transgene. However, the sequences necessary for expression comparable to that of MUC7 in human salivary glands may be missing from this construct. Western blot analysis of protein extracts from different tissues of transgenic mice line 3 showed that MUC7 gene product was produced in the submandibular-sublingual gland complex of both male and female mice and not in the other tissues examined.

Animals↗
PubMed1993

Transgenic mouse models of vasopressin expression.

Arginine vasopressin is a nine-amino acid neuropeptide hormone important in the regulation of water metabolism. It also may have a role in other physiological functions, such as blood pressure regulation and the response to stress. Whole animal studies have provided a good understanding of vasopressin physiology and regulation of the normal vasopressin gene, and in vitro cell culture studies have demonstrated important features of the intracellular regulation of vasopressin gene expression. Transgenic mice provide useful models for the study of the in vivo regulation of gene expression. Previously reported mouse lines transgenic with vasopressin gene constructs have not expressed the transgene in a tissue distribution similar to that detected for the endogenous mouse vasopressin gene. An 8.2-kb genomic construct of the rat vasopressin gene, including 3 kb each of 5' and 3' flanking sequences, has been used to develop a line of transgenic mice. These animals express the transgene in a tissue-specific manner, demonstrate appropriate osmotic regulation of transgenic vasopressin mRNA, and have normal water metabolism. Animals homozygous for the 8.2-kb transgene have increased basal plasma levels of vasopressin peptide but have no apparent change in basal water metabolism. The findings with this and other previously reported mouse lines transgenic for vasopressin constructs provide a basis for developing future transgenic lines to study the in vivo regulation of the vasopressin gene.

Animals↗
PubMed1995

Inheritance of gusA and neo genes in transgenic rice.

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T0) transformants and their progeny plants. T0 plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3') II] and the beta-glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S. Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T1 to T2 plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T2 generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.

Blotting, Southern↗
PubMed2003

Terpenoid metabolism in wild-type and transgenic Arabidopsis plants.

Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.

Acyclic Monoterpenes↗
PubMed2002

A novel animal model of thymic tumour: development of epithelial thymoma in transgenic rats carrying human T lymphocyte virus type I pX gene.

The pX region encodes a major product of human T lymphocyte virus type I (HTLV-I) that has been implicated previously in tumour formation. To investigate the pathogenesis of pX gene in lymphoid tissues, we established a series of novel transgenic rats carrying the pX gene under the control of a rat lymphocyte-specific protein tyrosine kinase (p56lck) proximal promoter. The transgene was constructed with the -269 to +26 region of a rat p56lck proximal promoter and the pX cDNA, and was microinjected into fertilized ova of Fischer 344/jcl female rats. Six transgenic lines from 114 pups were established. Integration and expression of the transgene were detected by polymerase chain reaction (PCR) and Southern hybridization or by reverse transcriptase-PCR, northern hybridization, and immunostaining. Thymic tumours with lethal expansion occurred in 4 of 6 transgenic lines. The tumour consisted of spindle shaped cells. Immunohistochemical and ultra-structural analysis characterized the tumour cells to as epithelial cell type, and in the tumour arose in the medulla. Therefore, the tumour is classified into predominantly epithelial and spindle cell of medullary thymoma (type A of the new World Health Organization classification), as based on the human classification. Tumor occurrence increased in proportion to levels of the pX transcription in the thymus, for each line, and sex distinction was evident regarding rates related to tumour expansion. The transgenic rat model described here is suitable as a model for analysing tumorigenesis in epithelial thymoma occurring in humans.

Animals↗