Showing papers on "Transgene published in 2002"
TL;DR: Transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter are generated and transgenic rats that express GFP at high levels are generated, suggesting that this technique can be used to produce other transgenic animal species.
Abstract: Single-cell mouse embryos were infected in vitro with recombinant lentiviral vectors to generate transgenic mice carrying the green fluorescent protein (GFP) gene driven by a ubiquitously expressing promoter. Eighty percent of founder mice carried at least one copy of the transgene, and 90% of these expressed GFP at high levels. Progeny inherited the transgene(s) and displayed green fluorescence. Mice generated using lentiviral vectors with muscle-specific and T lymphocyte–specific promoters expressed high levels of GFP only in the appropriate cell types. We have also generated transgenic rats that express GFP at high levels, suggesting that this technique can be used to produce other transgenic animal species.
2,051 citations
TL;DR: It is suggested that p53 has a role in regulating organismal ageing by generating mice with a deletion mutation in the first six exons of the p53 gene that express a truncated RNA capable of encoding a carboxy-terminal p53 fragment.
Abstract: The p53 tumour suppressor is activated by numerous stressors to induce apoptosis, cell cycle arrest, or senescence. To study the biological effects of altered p53 function, we generated mice with a deletion mutation in the first six exons of the p53 gene that express a truncated RNA capable of encoding a carboxy-terminal p53 fragment. This mutation confers phenotypes consistent with activated p53 rather than inactivated p53. Mutant (p53+/m) mice exhibit enhanced resistance to spontaneous tumours compared with wild-type (p53+/+) littermates. As p53+/m mice age, they display an early onset of phenotypes associated with ageing. These include reduced longevity, osteoporosis, generalized organ atrophy and a diminished stress tolerance. A second line of transgenic mice containing a temperature-sensitive mutant allele of p53 also exhibits early ageing phenotypes. These data suggest that p53 has a role in regulating organismal ageing.
1,387 citations
TL;DR: A transgenic mouse line is generated in which Cre-ER is ubiquitously expressed to permit temporally regulated Cre-mediated recombination in diverse tissues of the mouse at embryonic and adult stages and this inducible Cre system will be a broadly useful tool to modulate gene activity in mouse embryos, adults, and culture systems where temporal control is an important consideration.
1,311 citations
TL;DR: The results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone and the animal models devoid of or overexpressing the peptide represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hePCidin action.
Abstract: We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.
922 citations
TL;DR: Here a protocol is described to create gfp fusion constructs that are ready for injection into the C. elegans gonad within one day, with no need for subcloning procedures, and is schematically outlined in Figure 1.
Abstract: In the nematode Caenorhabditis elegans, gene expression patterns are most often determined by generating a fusion of the promoter of the gene under study to a reporter gene such as lacZ or gfp (3,4,7,8,11). Of the almost 20 000 genes in the worm, the spatial expression pattern of only a few hundred genes has been determined (http:// www.wormbase.org). If the reporter gene is fused to the full coding sequence of the gene under study, one can also obtain useful hints about the subcellular localization of the protein. Apart from revealing potential clues about the gene under study, reporter gene fusions serve as invaluable markers to assess the fate of individual cells in defined mutant backgrounds (1,13). The computational comparison of the promoter sequences of large numbers of co-expressed genes will also lead to a better understanding of the underlying logic of transcriptional control (14). Hence, the availability of a large number of reporter genes that provide gene expression information with temporal and spatial resolution is of significant interest in the post-genome era. A method to rapidly create reporter gene fusions on a large scale would thus be a desirable tool to have at hand. All current methods for generating reporter gene fusions encompass timeconsuming DNA subcloning and DNA purification protocols (3,4,8,11). Here a protocol is described to create gfp fusion constructs that are ready for injection into the C. elegans gonad within one day, with no need for subcloning procedures. The protocol is a modified version of previously described PCRbased fusions of overlapping DNA fragments (9,12) and is schematically outlined in Figure 1. The protocol entails a reaction in which two primary PCR products (Figure 1, product nos. 1 and 2) are fused by PCR with a set of nested primers. In PCR no. 1, the promoter (or the complete gene) under study is amplified from worm genomic DNA that was prepared by digestion with 60 μg/mL proteinase K (1 h lysis at 65oC; 15 min at 95oC inactivation; reaction buffer: 10 mM Tris-HCl, pH 8.2, 50 mM KCl, 2.5 mM MgCl2, 0.45% Nonidet P-40, 0.45% Tween 20, 0.01% gelatin) or, alternatively and often more efficiently, from a preparation of cosmid DNA. In the parallel PCR no. 2, the gfp coding sequence plus the generically used 3′ untranslated region (UTR) from the unc-54 gene are amplified from the standard Fire vector pPD95.75 (http://www.ciwemb.edu/pages/firelab.html) (4). The 3′ primer for the promoter/gene (Figure 1, termed “B”) has a 24-nucleotide overhang to the gfp vector pPD95.75. If product no. 1 contains the coding region of the gene of interest, then it is important to ensure with primer B that the gfp is fused in frame to the gene of interest. Primer “C” does not need to have an overlap to the specific promoter/gene to be amplified, thus making C a generic primer that can be used for every fusion reaction. Gel purification of the two primary products was often found to inhibit the fusion PCR for unknown reasons. The two primary products are thus used with no further purification for the fusion PCR, which further speeds up the whole process. The concentration of the product # 1 and # 2 is roughly estimated by agarose gel electrophoresis, and an aliquot of the reaction is then diluted with water to 10–50 ng/μL each product. In case the yield of the PCR product is less than 10 ng/μL, it can also be used undiluted; we have even encountered cases in which the first PCR product was invisible on a gel and, nevertheless, obtained a fusion product. After this estimation and dilution step, 1 μL each diluted (or undiluted) reaction is used in the fusion PCR. For this reaction, it is obligatory to use nested primers (Figure 1, A* and D*). Although in most cases one will get a single band from the PCR fusion, another band can occasionally be seen, possibly a gfp dimer; sometimes this additional band may be much stronger than the desired PCR fusion product. This band can be ignored and considered as some sort of “carrier DNA” for the ensuing microinjection into the worms. The concentration of product no. 3 is estimated by agarose gel electrophoresis, and the reaction is diluted Benchmarks
662 citations
TL;DR: A method for efficient in vivo delivery of siRNAs to organs of postnatal mice is described and effective and specific inhibition of transgene expression in a variety of organs is demonstrated.
Abstract: It has recently been shown that RNA interference can be induced in cultured mammalian cells by delivery of short interfering RNAs (siRNAs). Here we describe a method for efficient in vivo delivery of siRNAs to organs of postnatal mice and demonstrate effective and specific inhibition of transgene expression in a variety of organs.
661 citations
TL;DR: Somatic gene transfer is a promising therapeutic strategy, but it may also evoke new types of side effects related to genetic damage or transgene activity.
Abstract: Somatic gene transfer is a promising therapeutic strategy, but it may also evoke new types of side effects related to genetic damage or transgene activity. Retroviral vectors, the best tool currently available for stable genetic modification, integrate at random positions in the cellular genome. The
653 citations
TL;DR: It is reported that FGF19 transgenic mice had a significant and specific reduction in fat mass that resulted from an increase in energy expenditure and did not become obese or diabetic on a high fat diet.
Abstract: The fibroblast growth factors (FGFs), and the corresponding receptors, are implicated in more than just the regulation of epithelial cell proliferation and differentiation. Specifically, FGF23 is a regulator of serum inorganic phosphate levels, and mice deficient in FGF receptor-4 have altered cholesterol metabolism. The recently described FGF19 is unusual in that it is nonmitogenic and appears to interact only with FGF receptor-4. Here, we report that FGF19 transgenic mice had a significant and specific reduction in fat mass that resulted from an increase in energy expenditure. Further, the FGF19 transgenic mice did not become obese or diabetic on a high fat diet. The FGF19 transgenic mice had increased brown adipose tissue mass and decreased liver expression of acetyl coenzyme A carboxylase 2, providing two mechanisms by which FGF19 may increase energy expenditure. Consistent with the reduction in expression of acetyl CoA carboxylase 2, liver triglyceride levels were reduced.
572 citations
TL;DR: The findings suggest that long-term exposure of the brain to proinflammatory mediators such as IL-6, as is seen in certain degenerative disorders and infections, can interfere with adult neurogenesis.
Abstract: Postnatal neurogenesis can be modulated after brain injury, but the role of the attendant expression of inflammatory mediators in such responses remains to be determined. Here we report that transgenically directed production of interleukin-6 (IL-6) by astroglia decreased overall neurogenesis by 63% in the hippocampal dentate gyrus of young adult transgenic mice. The proliferation, survival, and differentiation of neural progenitor cells labeled with the thymidine analog bromodeoxyuridine were all reduced in the granule cell layer of these mice, whereas their distribution and gliogenesis appeared normal. These effects were not a consequence of general toxicity of the IL-6 transgene, because they were manifested in the absence of neuronal death and of major changes in glial cell number and morphology. These findings suggest that long-term exposure of the brain to proinflammatory mediators such as IL-6, as is seen in certain degenerative disorders and infections, can interfere with adult neurogenesis.
570 citations
TL;DR: The results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.
Abstract: Formation of somatic embryos in plants is known to require high concentrations of auxin or 2,4-dichlorophenoxyacetic acid (2,4-D), which presumably acts to trigger a signalling cascade. However, very little is known about the molecular mechanism that mediates the vegetative-to-embryogenic transition. We have employed a genetic approach to dissect the signal transduction pathway during somatic embryogenesis. In a functional screen using a chemical-inducible activation-tagging system, we identified two alleles of Arabidopsis gene PGA6 whose induced overexpression caused high-frequency somatic embryo formation in all tissues and organs tested, without any external plant hormones. Upon inducer withdrawal, all these somatic embryos were able to germinate directly, without any further treatment, and to develop into fertile adult plants. PGA6 was found to be identical to WUSCHEL (WUS), a homeodomain protein previously shown to be involved in specifying stem cell fate in shoot and floral meristems. Transgenic plants carrying an estradiol-inducible XVE-WUS transgene can phenocopy pga6-1 and pga6-2. Our results suggest that WUS/PGA6 also plays a key role during embryogenesis, presumably by promoting the vegetative-to-embryogenic transition and/or maintaining the identity of the embryonic stem cells.
556 citations
TL;DR: Lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos and ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.
Abstract: The introduction of foreign genes into early mouse embryos and embryonic stem (ES) cells is invaluable for the analysis of gene function and regulation in the living animal. The use of vectors derived from retroviruses as gene transfer vehicles in this setting has had limited success because of silencing of transgene expression. Here, we show that vectors derived from lentiviruses, which are complex retroviruses, can efficiently deliver genes to murine ES cells and that transgene expression is stable during proliferation of undifferentiated ES cells. The transgene is expressed during differentiation of ES cells in vitro (embryoid bodies) and in vivo (teratomas). Transfer of lentivector-transduced ES cells into blastocysts resulted in chimeric animals that expressed the transgene in multiple tissues. Embryos derived from crossings of chimeric mice expressed the transgene, indicating successful germ-line transmission. Infection of murine preimplantation embryos at morula stage with lentiviral vectors resulted in stable transduction and expression of the transgene in mouse embryos and in newborn mice. Finally, human ES cells were transduced by lentiviral vectors and expressed the transgene over several passages. Thus, lentiviral vectors represent a significant improvement over oncoretroviral vectors used previously for gene transfer into murine ES cells and preimplantation embryos. Ability to transfer foreign genes into human ES cells has potential relevance for the development of gene and cell-based therapies.
TL;DR: Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules.
Abstract: We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a single HsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules.
TL;DR: The generation and characterization of mice carrying supernumerary copies of the p53 gene in the form of large genomic transgenes are reported, proving that cancer resistance can be enhanced by a simple genetic modification and in the absence of undesirable effects.
Abstract: The tumor suppressor p53 is critical in preventing cancer due to its ability to trigger proliferation arrest and cell death upon the occurrence of a variety of stresses, most notably, DNA damage and oncogenic stress. Here, we report the generation and characterization of mice carrying supernumerary copies of the p53 gene in the form of large genomic transgenes. Prior to this, we demonstrate that the p53 transgenic allele (p53-tg), when present in a p53-null genetic background, behaves as a functional replica of the endogenous gene. ‘Super p53’ mice, carrying p53-tg alleles in addition to the two endogenous alleles, exhibit an enhanced response to DNA damage. Importantly, ‘super p53’ mice are significantly protected from cancer when compared with normal mice. Finally, in contrast to previously reported mice with constitutively active p53, ‘super p53’ mice do not show any indication of premature aging, probably reflecting the fact that p53 is under normal regulatory control. Together, our results prove that cancer resistance can be enhanced by a simple genetic modification and in the absence of undesirable effects.
TL;DR: Constitutive expression of viral proteins leads to common pathologic features of hepatitis C in the absence of specific anti-viral immune responses, while additional low level expression of nonstructural proteins increases the risk of cancer.
TL;DR: Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway, and the activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diph phosphate synthase, and incorporation of isopentanyl dphosphate into phy toene) were not significantly altered.
Abstract: Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.
TL;DR: The recently developed testis cell transplantation method provides a powerful approach to studying the biology of the male germline stem cell and its microenvironment, the stem cell niche.
Abstract: The recently developed testis cell transplantation method provides a powerful approach to studying the biology of the male germline stem cell and its microenvironment, the stem cell niche. The technique also is being used to examine spermatogenic defects, correct male infertility, and generate transgenic animals.
TL;DR: The results indicate that PON1 protects against atherosclerosis in a dose-dependent manner and suggest that it may be a potential target for developing therapeutic agents for the treatment of cardiovascular disease.
Abstract: Background— Serum paraoxonase (PON1), an enzyme carried on HDL, inhibits LDL oxidation, and in human population studies, low PON1 activity is associated with atherosclerosis. In addition, PON1 knockout mice are more susceptible to lipoprotein oxidation and atherosclerosis. To evaluate whether PON1 protects against atherosclerosis and lipid oxidation in a dose-dependent manner, we generated and studied human PON1 transgenic mice. Methods and Results— Human PON1 transgenic mice were produced by using bacterial artificial chromosome genomic clones. The mice had 2- to 4-fold increased plasma PON1 levels, but plasma cholesterol levels were unchanged. Atherosclerotic lesions were significantly reduced in the transgenic mice when both dietary and apoE-null mouse models were used. HDL isolated from the transgenic mice also protected against LDL oxidation more effectively. Conclusions— Our results indicate that PON1 protects against atherosclerosis in a dose-dependent manner and suggest that it may be a potential ...
TL;DR: The Sox2Cre line is likely to be more effective in generating complete, epiblast-specific removal of gene activity, and the mosaic activity of the MORE line will be helpful in generating partial loss-of-function phenotypes in the embryo-proper.
TL;DR: Results from the current study suggest that heterologous CBF1expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.
Abstract: In an attempt to improve stress tolerance of tomato (Lycopersicon esculentum) plants, an expression vector containing an Arabidopsis C-repeat/dehydration responsive element binding factor 1 (CBF1) cDNA driven by a cauliflower mosaic virus 35S promoter was transferred into tomato plants. Transgenic expression of CBF1 was proved by northern- and western-blot analyses. The degree of chilling tolerance of transgenic T1 and T2 plants was found to be significantly greater than that of wild-type tomato plants as measured by survival rate, chlorophyll fluorescence value, and radical elongation. The transgenic tomato plants exhibited patterns of growth retardation; however, they resumed normal growth after GA3 (gibberellic acid) treatment. More importantly, GA3-treated transgenic plants still exhibited a greater degree of chilling tolerance compared with wild-type plants. Subtractive hybridization was performed to isolate the responsive genes of heterologous Arabidopsis CBF1 in transgenic tomato plants. CATALASE1 (CAT1) was obtained and showed activation in transgenic tomato plants. The CAT1 gene and catalase activity were also highly induced in the transgenic tomato plants. The level of H2O2 in the transgenic plants was lower than that in the wild-type plants under either normal or cold conditions. The transgenic plants also exhibited considerable tolerance against oxidative damage induced by methyl viologen. Results from the current study suggest that heterologous CBF1 expression in transgenic tomato plants may induce several oxidative-stress responsive genes to protect from chilling stress.
TL;DR: Results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.
Abstract: A DNA cassette containing an Arabidopsis C repeat/dehydration-responsive element binding factor 1 (CBF1) cDNA and a nos terminator, driven by a cauliflower mosaic virus 35S promoter, was transformed into the tomato (Lycopersicon esculentum) genome. These transgenic tomato plants were more resistant to water deficit stress than the wild-type plants. The transgenic plants exhibited growth retardation by showing dwarf phenotype, and the fruit and seed numbers and fresh weight of the transgenic tomato plants were apparently less than those of the wild-type plants. Exogenous gibberellic acid treatment reversed the growth retardation and enhanced growth of transgenic tomato plants, but did not affect the level of water deficit resistance. The stomata of the transgenic CBF1 tomato plants closed more rapidly than the wild type after water deficit treatment with or without gibberellic acid pretreatment. The transgenic tomato plants contained higher levels of Pro than those of the wild-type plants under normal or water deficit conditions. Subtractive hybridization was used to isolate the responsive genes to heterologous CBF1 in transgenic tomato plants and the CAT1 (CATALASE1) was characterized. Catalase activity increased, and hydrogen peroxide concentration decreased in transgenic tomato plants compared with the wild-type plants with or without water deficit stress. These results indicated that the heterologous Arabidopsis CBF1 can confer water deficit resistance in transgenic tomato plants.
TL;DR: Demonstration of INSL3 as the ligand for an orphan G protein-coupled receptor based on common phenotypes of ligand and receptor null mice facilitates understanding of the mechanism of testis descent and allows studies on the role of IN SL3 in gonadal and other physiological processes.
TL;DR: An in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation is described and it is found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of the approach.
Abstract: Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.
TL;DR: By applying the mammalian codon usage to Cre recombinase, Cre expression was improved, as determined by immunoblot and functional analysis, in three different mammalian cell lines, and it was indicated that iCre should facilitate genetic experiments in the mouse.
Abstract: By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.
TL;DR: In this paper, the formation of hepatocellular carcinoma in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle was described.
Abstract: Most mouse models of hepatocellular carcinoma have expressed growth factors and oncogenes under the control of a liver-specific promoter. In contrast, we describe here the formation of liver tumors in transgenic mice overexpressing human fibroblast growth factor 19 (FGF19) in skeletal muscle. FGF19 transgenic mice had elevated hepatic α-fetoprotein mRNA as early as 2 months of age, and hepatocellular carcinomas were evident by 10 months of age. Increased proliferation of pericentral hepatocytes was demonstrated by 5-bromo-2′-deoxyuridine incorporation in the FGF19 transgenic mice before tumor formation and in nontransgenic mice injected with recombinant FGF19 protein. Areas of small cell dysplasia were initially evident pericentrally, and dysplastic/neoplastic foci throughout the hepatic lobule were glutamine synthetase-positive, suggestive of a pericentral origin. Consistent with chronic activation of the Wingless/Wnt pathway, 44% of the hepatocellular tumors from FGF19 transgenic mice had nuclear staining for β-catenin. Sequencing of the tumor DNA encoding β-catenin revealed point mutations that resulted in amino acid substitutions. These findings suggest a previously unknown role for FGF19 in hepatocellular carcinomas.
TL;DR: For both MnSOD and Cu/ZnSOD lines, increased life span was not associated with decreased metabolic activity, as measured by O2 consumption, and was consistent with previous observations that catalase is present in excess in the adult fly with regard to life span.
Abstract: A transgenic system ("FLP-out") based on yeast FLP recombinase allowed induced overexpression of MnSOD enzyme in adult Drosophila melanogaster. With FLP-out a brief heat pulse (HP) of young, adult flies triggered the rearrangement and subsequent expression of a MnSOD transgene throughout the adult life span. Control (no HP) and overexpressing (HP) flies had identical genetic backgrounds. The amount of MnSOD enzyme overexpression achieved varied among six independent transgenic lines, with increases up to 75%. Life span was increased in proportion to the increase in enzyme. Mean life span was increased by an average of 16%, with some lines showing 30-33% increases. Maximum life span was increased by an average of 15%, with one line showing as much as 37% increase. Simultaneous overexpression of catalase with MnSOD had no added benefit, consistent with previous observations that catalase is present in excess in the adult fly with regard to life span. Cu/ZnSOD overexpression also increases mean and maximum life span. For both MnSOD and Cu/ZnSOD lines, increased life span was not associated with decreased metabolic activity, as measured by O2 consumption.
TL;DR: A cluster of WRKY binding sites in the promoter of the gene are identified and demonstrated that they acted as negative regulatory elements for the inducible expression of AtWRKY18, which potentiated developmentally regulated defense responses in transgenic plants without causing substantial negative effects on plant growth.
Abstract: AtWRKY18 is a pathogen- and salicylic acid-induced Arabidopsis transcription factor containing the plant-specific WRKY zinc finger DNA-binding motif. In the present study, we have transformed Arabidopsis plants with AtWRKY18 under control of the cauliflower mosaic virus 35S promoter. Surprisingly, transgenic plants expressing high levels of AtWRKY18 were stunted in growth. When expressed at moderate levels, AtWRKY18 potentiated developmentally regulated defense responses in transgenic plants without causing substantial negative effects on plant growth. As they grew from seedling to mature stages, transgenic AtWRKY18 plant showed marked increase in the expression of pathogenesis-related genes and resistance to the bacterial pathogen Pseudomonas syringae, whereas wild-type plants exhibited little enhancement in these defense responses. Potentiation of developmentally regulated defense responses by AtWRKY18 was not associated with enhanced biosynthesis of salicylic acid but required the disease resistance regulatory protein NPR1/NIM1. Thus, AtWRKY18 can positively modulate defense-related gene expression and disease resistance. To study the regulated expression of AtWRKY18, we have identified a cluster of WRKY binding sites in the promoter of the gene and demonstrated that they acted as negative regulatory elements for the inducible expression of AtWRKY18. These negative cis-acting elements may prevent overexpression of AtWRKY18 during the activation of plant defense responses that could be detrimental to plant growth as inferred from the transgenic plants ectopically expressing the transgene.
TL;DR: Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage, and PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ.
Abstract: Transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the mouse myelin proteolipid protein (PLP) gene promoter have been developed to investigate cells in the oligodendrocyte lineage. Transgene expression is consistent with the developmental expression of PLP, with cells at all stages of oligodendrocyte differentiation clearly visualized. These animals were analyzed to establish the time course of oligodendrocyte progenitor migration, proliferation, and differentiation in neonatal cortex. In these animals, two populations of NG2 proteoglycan-positive oligodendrocyte progenitor cells were identified that exist in postnatal subventricular zone and cortex. These two populations are distinguished by the presence or absence of PLP gene expression. Thus, PLP gene expression defines a subpopulation of NG2-positive cells from very early postnatal ages, which migrates toward the pial surface and proliferates in situ . EGFP + /NG2 + cells are present in the cortex from postnatal day 1, and they remain in the cortex as undifferentiated oligodendrocyte progenitors for up to 3 weeks before myelination begins. These data could be explained by the presence of an important inhibitor of oligodendrocyte differentiation in the cortex during this period, which is downregulated in a region-specific manner to allow myelination. On the other hand, it is possible that oligodendrocyte progenitor cells remain undifferentiated in cortex until an essential signal is produced in situ to induce differentiation.
TL;DR: The induced ovarian tumors in mice resembled human ovarian carcinomas in their rapid progression and intraperitoneal metastatic spread and it was demonstrated that the ovarian surface epithelium is the precursor tissue for these ovarian carcinoma, and that introduction of oncogenes causes phenotypic changes in the ovariansurface epithelial cells.
TL;DR: To determine the impact of tumor progression on the reversibility of Neu-induced tumorigenesis, the tetracycline regulatory system is used to conditionally express activated Neu in the mammary epithelium of transgenic mice.
TL;DR: The speed and specificity of inhibition suggests that myosin-1c participates in adaptation in hair cells, the sensory cells of the inner ear.