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At least 991 records · Page 55Linked to original sources
PubMed1993

Increased production of cadaverine and anabasine in hairy root cultures of Nicotiana tabacum expressing a bacterial lysine decarboxylase gene.

Several hairy root cultures of Nicotiana tabacum varieties, carrying two direct repeats of a bacterial lysine decarboxylase (ldc) gene controlled by the cauliflower mosaic virus (CaMV) 35S promoter expressed LDC activity up to 1 pkat/mg protein. Such activity was, for example, sufficient to increase cadaverine levels of the best line SR3/1-K1,2 from ca. 50 micrograms (control cultures) to about 700 micrograms/g dry mass. Some of the overproduced cadaverine of this line was used for the formation of anabasine, as shown by a 3-fold increase of this alkaloid. In transgenic lines with lower LDC activity the changes of cadaverine and anabasine levels were correspondingly lower and sometimes hardly distinguishable from controls. Feeding of lysine to root cultures, even to those with low LDC activity, greatly enhanced cadaverine and anabasine levels, while the amino acid had no or very little effect on controls and LDC-negative lines.

Anabasine↗
PubMed2003

Transgene instability in mice injected with an in vitro methylated Igf2 gene.

Foreign DNA injected into mouse embryos integrates into the host chromosomes and is usually transmitted stably to the progeny. Rare cases of transgene instability have been described, and these can help our understanding of the rules that govern the organization and stability of endogenous DNA. We have observed unusual inheritance in three transgenic lines produced with a partially in vitro methylated Igf2 construct. All three founders transmitted to their progeny two different transgene patterns, A and B. Pattern A was inherited in accordance with expectation, whereas pattern B was associated with several abnormal characteristics, including fewer than expected transgenic progeny, evidence for instability and loss from the somatic tissues of some of the progeny, and high incidence of runting and perinatal death that did not appear correlated with transgene retention. The absence of these features in transgenic mice produced with the unmethylated version of the same construct indicated that prior methylation played a role in the unusual behavior of these transgenes. We hypothesize that patterns A and B were formed by transgenes that differed in their methylation, and that pattern B methylation led to instability of the transgene locus. Runting and early lethality in the pattern B sublines may be the result of transgene rearrangements, which result in transgene amplification with adverse effects of increased IGFII dosage, and/or deletions, which may affect endogenous genes required for viability. These findings provide further evidence that DNA methylation plays a role in genome stability and indicate that perturbations in the normal pattern of methylation may have destabilizing effects that extend through several generations.

Animals↗
PubMed2001

Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans.

A genomic copy of a gut-expressed Haemonchus contortus candidate vaccine antigen, pepsinogen, was isolated using the polymerase chain reaction (PCR). The isolated sequence was 4 kb in length and contained eight introns ranging in size from 54 to 1475 base pairs. This sequence, together with its 3' non-coding DNA region containing a polyadenylation signal sequence, was cloned into the Bluescript SK(+) vector immediately downstream of the Caenorhabditis elegans cpr-5 gene promoter. This promoter has been shown previously to direct protein expression to the gut of C. elegans. The construct was micro-injected into DR96 unc-76(e911) mutant C. elegans together with a rescue plasmid and transgenic worms identified by reversion back to wild-type phenotype. Two transgenic lines of C. elegans were established. The presence of the injected construct and of the Haemonchus pepsinogen transcript in transgenic worms was confirmed by PCR analysis. Correct splicing of intronic sequences was observed. Immunohistochemistry showed expression of the Haemonchus pepsinogen protein in the gut of transgenic C. elegans, with reactivity evident in the larval and adult stages. Expression of the Haemonchus pepsinogen in C. elegans affirms the role of C. elegans as a model for parasitic nematodes and demonstrates its potential as a vector for expression of candidate vaccine antigens from parasitic nematodes.

Animals↗
PubMed2001

Co-suppression mediated virus resistance in transgenic tobacco plants harboring the 3'-untranslated region of Andean potato mottle virus.

Nicotiana tabacum plants were transformed with the 3'-untranslated region of the Andean Potato Mottle Virus (APMoV) genome RNA-2. Three strategies were used: the introduction of this region in sense and in antisense orientation and of a fragment comprising the entire 3'-untranslated region from RNA-2 and part of the CP22 coat protein sequence. The transgenic lines were inoculated with the virus and different responses were observed, ranging from susceptibility to APMoV to complete immunity to virus infection, in which neither the virion nor viral RNA was detected in the inoculated leaf and leaves that emerged after inoculation. The R1 progeny from different R0 lines also showed an array of virus resistance phenotypes, which was not correlated with the zygotic state of the transgene. Resistance was positively correlated with low levels of transgene mRNA accumulation, indicating a co-suppression-mediated mechanism towards the transgenic transcripts and APMoV viral RNA.

3' Untranslated Regions↗
PubMed1999

Murine MHC class II locus control region drives expression of human beta-glucocerebrosidase in antigen presenting cells of transgenic mice.

Gaucher disease is the most prevalent lysosomal storage disorder in humans, resulting from an inherited deficiency of the enzyme glucocerebrosidase. Although the enzyme is ubiquitously expressed, cells of the reticuloendothelial system are particularly affected since they accumulate the undigested glucosylceramide substrate through their role in scavenging and breaking down cell debris. Gaucher disease is an attractive target for somatic gene therapy. To test the ability to express the enzyme in the affected cell types we have generated transgenic mice expressing human glucocerebrosidase under the control of the murine major histocompatibility complex (MHC) class II Ead locus control region (LCR). The four transgenic lines express the human enzyme in a copy number-dependent manner, independent of the integration site of the transgene. Over-expression of the human enzyme in mice did not result in any abnormal phenotype or pathology during the period of observation (> 2 years). The enzyme is expressed in B cells, monocytes, dendritic cells, thymic epithelial cells, and macrophages in various tissues: the peritoneal cavity, bone marrow, spleen, kidney, gastrointestinal tract, Kupffer cells in the liver and alveolar macrophages in lungs. Expression in the brain was limited to perivascular macrophages and was not seen in microglial cells. Therefore, the MHC class II LCR could potentially be of use in somatic gene therapy for type 1 Gaucher disease.

Animals↗
PubMed2003

Overexpression of a stress-inducible aldehyde dehydrogenase gene from Arabidopsis thaliana in transgenic plants improves stress tolerance.

In plants, oxidative stress is one of the major causes of damage as a result of various environmental stresses. Oxidative stress is primarily because of the excessive accumulation of reactive oxygen species (ROS). The amplification of ROS damage is further stimulated by the accumulation of toxic degradation products, i.e. aldehydes, arising from reactions of ROS with lipids and proteins. Previously, the isolation of dehydration-inducible genes encoding aldehyde dehydrogenases (ALDHs) was reported from the desiccation-tolerant plant Craterostigma plantagineum and Arabidopsis thaliana. ALDHs belong to a family of NAD(P)+-dependent enzymes with a broad substrate specificity that catalyze the oxidation of various toxic aldehydes to carboxylic acids. Analysis of transcript accumulation revealed that Ath-ALDH3 is induced in response to NaCl, heavy metals (Cu2+ and Cd2+), and chemicals that induce oxidative stress (methyl viologen (MV) and H2O2). To investigate the physiological role and possible involvement of ALDHs in stress protection, we generated transgenic Arabidopsis plants overexpressing Ath-ALDH3. Transgenic lines show improved tolerance when exposed to dehydration, NaCl, heavy metals (Cu2+ and Cd2+), MV, and H2O2. Tolerance of transgenic plants is correlated with decreased accumulation of lipid peroxidation-derived reactive aldehydes (as measured by malondialdehyde) compared to wild-type plants. Increased activity of Ath-ALDH3 appears to constitute a detoxification mechanism that limits aldehyde accumulation and oxidative stress, thus revealing a novel pathway of detoxification in plants. We suggest that Ath-ALDH3 could be used to obtain plants with tolerance to diverse environmental stresses.

Adaptation, Physiological↗
PubMed1996

Full genetic rescue of adenosine deaminase-deficient mice through introduction of the human gene.

We have shown recently that adenosine deaminase (ADA)-deficient mice die perinatally with severe liver cell degeneration. In addition to enzyme substitution, we report the restoration of viability through introduction of the human ADA gene. The ADA gene is subject to complex developmental and tissue-specific regulation. To include the cis-regulatory elements necessary for correct regulation of the human ADA gene, a large transgenic locus constituting the human ADA gene with 10 kb of 5' and 4 kb of 3' flanking sequences was generated by co-injection of two overlapping DNA fragments into murine zygotes. Probably as a result of extrachromosomal (homologous) recombination between the fragments, one of the two transgenic lines contained a reconstituted, functional human ADA gene. As in man, human ADA expression generally was low in these transgenic mice, but high in the thymus, spleen and gastro-duodenal part of the gut. Apparently, all cis-regulatory elements essential for a human expression pattern were incorporated in the transgene and were functional in the murine background. Similarly to man, the upper alimentary tract of the transgenic mice revealed low human ADA activity in contrast to extremely high levels of murine ADA. The human gene probably lacks the cis-regulatory elements that target high level murine ADA expression to the murine upper alimentary tract. ADA-deficient mice rescued by introduction of the human ADA transgene appeared histologically and immunologically normal. Apparently, human ADA can complement murine ADA in all tissues, even in the epithelium of the upper alimentary tract where human ADA activity is as much as 70-fold lower than murine ADA activity in wild-type mice. Clearly, the lethal phenotype of ADA-deficient mice is due to the absence of ADA.

Adenosine Deaminase↗
PubMed2004

Untranslated regions from C4 amaranth AhRbcS1 mRNAs confer translational enhancement and preferential bundle sheath cell expression in transgenic C4 Flaveria bidentis.

Many aspects of photosynthetic gene expression are posttranscriptionally regulated in C4 plants. To determine if RbcS mRNA untranslated regions (UTRs) in themselves could confer any characteristic C4 expression patterns, 5'- and 3'-UTRs of AhRbcS1 mRNA from the C4 dicot amaranth were linked to a gusA reporter gene. These were constitutively transcribed from a cauliflower mosaic virus promoter and assayed for posttranscriptional expression patterns in transgenic lines of the C4 dicot Flaveria bidentis. Three characteristic C4 expression patterns were conferred by heterologous AhRbcS1 UTRs in transgenic F. bidentis. First, the AhRbcS1 UTRs conferred strong translational enhancement of gusA expression, relative to control constructs lacking these UTRs. Second, while the UTRs did not appear to confer tissue-specific expression when analyzed by beta-glucuronidase activity assays, differences in gusA mRNA accumulation were observed in leaves, stems, and roots. Third, the AhRbcS1 UTRs conferred preferential gusA expression (enzyme activity and gusA mRNA accumulation) in leaf bundle sheath cells. AhRbcS1 UTR-mediated translational enhancement was also observed in transgenic C3 plants (tobacco [Nicotiana tabacum]) and in in vitro translation extracts. These mRNAs appear to be translated with different efficiencies in C4 versus C3 plants, indicating that processes determining overall translational efficiency may vary between these two categories of higher plants. Our findings suggest that the AhRbcS1 5'-UTR functions as a strong translational enhancer in leaves and other tissues, and may work synergistically with the 3'-UTR to modulate overall levels of Rubisco gene expression in different tissues and cell types of C4 plants.

Amaranthus↗
PubMed1998

Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis.

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C19-GAs. All bioactive GAs are C19-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA1, GA9, and GA20 were detected in seedlings of the transgenic line examined. GA4, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype.

Arabidopsis↗
PubMed2003

Modulation of medullary thyroid carcinoma penetrance suggests the presence of modifier genes in a RET transgenic mouse model.

We described previously a thyroid phenotype for transgenic mice expressing an activated Ret oncogene driven from a human calcitonin promoter. Medullary thyroid carcinomas (MTC), a tumor of the thyroid parafollicular C cells, occur in this transgenic line with a pathology analogous to that seen in patients with multiple endocrine neoplasia type 2 (MEN2). When the transgene was introgressed onto four different genetic backgrounds, between 0 and 98% of transgenic progeny developed thyroid tumors by 10 months of age, indicating that tumor penetrance could be modulated by genetic background. Furthermore, tumors on the CBA/ca and C57BL/6J backgrounds were significantly larger than those arising in BALB/c transgenic mice. These results are relevant to human MEN2 disease, because this model system may be used to study genes modifying thyroid tumor penetrance in the dominantly inherited human cancer syndrome.

Animals↗
PubMed2002

Double jeopardy: both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive.

In plants, the role of mitogen-activated protein kinase (MAPK) in reactive oxygen species (ROS)-based signal transduction processes is elusive. Despite the fact that ROS can induce MAPK activation, no direct genetic evidence has linked ROS-induced MAPK activation with the hypersensitive response, a form of programmed cell death. In tobacco, the major ROS-induced MAPK is salicylate-induced protein kinase (SIPK). We found through gain-of-function and loss-of-function approaches that both overexpression and RNA interference-based suppression of SIPK render the plant sensitive to ROS stress. Transgenic lines overexpressing a nonphosphorylatable version of SIPK were not ROS sensitive. Analysis of the MAPK activation profiles in ROS-stressed transgenic and wild-type plants revealed a striking interplay between SIPK and another MAPK (wound-induced protein kinase [WIPK]) in the different kinotypes. During continuous ozone exposure, abnormally prolonged activation of SIPK was seen in the SIPK-overexpression genotype, without WIPK activation, whereas strong and stable activation of WIPK was observed in the SIPK-suppressed lines. Thus, one role of activated SIPK in tobacco cells upon ROS stimulation appears to be control of the inactivation of WIPK.

Gene Expression Regulation, Enzymologic↗
PubMed2006

Generation and characterization of brain lipid-binding protein promoter-based transgenic mouse models for the study of radial glia.

Radial glia play an essential role in the generation of the cerebral cortex through their function as neuronal precursors and as neuronal migration guides. A molecular marker for radial glia in the developing central nervous system is the brain lipid-binding protein (BLBP). To generate mouse models for the visualization and study of radial glia, we expressed EGFP, EYFP, or dsRed2 in transgenic mice under the control of the BLBP promoter. In these transgenic lines, fluorescent protein expression is restricted to radial glia in the embryonic cortex and to astrocytes in the adult brain. Electroporation of the transgenes into embryonic cortex also resulted in radial glia-specific transgene expression. These BLBP promoter driven transgenic mice and organotypic brain slices expressing different fluorescent markers in a radial glia-specific manner will be useful tools to further study the differentiation and function of radial glia in distinct regions of the developing CNS.

Animals↗
PubMed2006

Nestin-Cre mediated deletion of Pitx2 in the mouse.

Nestin-Cre mice are widely used to generate gene deletions in the developing brain. Surprisingly, fewNestin-Cre lines have been characterized for their temporal and brain region-specific recombination. In addition, some Nestin-Cre lines express Cre outside the central nervous system, making it difficult to choose appropriate lines for targeting genes with brain region-restricted expression. Here we describe the properties of a Nestin-Cre transgenic line and its use for conditional deletions of Pitx2, a paired-like homeodomain transcription factor. We report that Nestin-Cre conditional Pitx2 mutant mice have ocular and craniofacial defects consistent with the role of human PITX2 in Rieger syndrome. Conditional mutants exhibit defects in midbrain neuronal development similar to those in Pitx2 homozygous null embryos, but lack the abnormalities in subthalamic nucleus neurons that occur with complete loss of Pitx2 function. These data indicate that normal differentiation of midbrain neurons depends upon adequate Pitx2 function during the period of active neurogenesis.

Animals↗
PubMed1998

A transcription activation system for regulated gene expression in transgenic plants.

A widely applicable promoter system is described that allows a gene of interest to be activated in specific plant tissues after a cross between defined transgenic lines. The promoter, pOp, consists of lac operators cloned upstream of a minimal promoter. No expression was detected from this promoter when placed upstream of a beta-glucuronidase (GUS) reporter gene in transgenic plants. Transcription from the promoter was activated by crossing reporter plants with activator lines that expressed a chimeric transcription factor, LhG4. This factor comprised transcription-activation domain-II from Gal4 of Saccharomyces cerevisiae fused to a mutant lac-repressor that binds its operator with increased affinity. When LhG4 was expressed from the CaMV 35S promoter, the spatial and quantitative expression characteristics of the 35S promoter were exhibited by the GUS reporter. The LhG4/pOp system may be used to study toxic or deleterious gene products, to coordinate the expression of multiple gene products, to restrict transgene phenotypes to the F1 generation, and to generate hybrid seed. The LhG4 system offers spatially regulated gene expression in the tissues of whole plants growing under normal conditions without the need for external intervention. It complements inducible expression systems that offer temporal control of gene expression in tissues that can be treated with inducing chemicals.

Bacterial Proteins↗
PubMed1998

Hyperinnervation of neuromuscular junctions caused by GDNF overexpression in muscle.

Overexpression of glial cell line-derived neurotrophic factor (GDNF) by muscle greatly increased the number of motor axons innervating neuromuscular junctions in neonatal mice. The extent of hyperinnervation correlated with the amount of GDNF expressed in four transgenic lines. Overexpression of GDNF by glia and overexpression of neurotrophin-3 and neurotrophin-4 in muscle did not cause hyperinnervation. Thus, increased amounts of GDNF in postsynaptic target cells can regulate the number of innervating axons.

Animals↗
PubMed2003

The VpreB1 enhancer drives developmental stage-specific gene expression in vivo.

In adult mice, the VpreB genes are expressed in bone marrow progenitor (pro-) and precursor (pre-) B cells. As part of the pre-B cell receptor, the proteins are crucial for the proliferation of these cells and consequently normal B lymphocyte development. Using cell lines, we identified a lineage- and developmental-stage-specific VpreB1 enhancer. Here, we analyze its specificity in vivo by generating transgenic mice in which expression of a reporter gene (human CD122) is regulated by the VpreB1 enhancer in the context of its own promoter. All transgenic lines expressed the reporter gene in the bone marrow in a copy number-independent manner, whereas expression levels were integration site-dependent. While the enhancer is not tissue specific, within the B cell lineage the expression pattern of human CD122 mimicked that of endogenous VpreB1. Thus, low levels were detected in pro-B cells, high levels in pre-BI and slightly lower levels in pre-BII cells; no expression was detected in immature/mature B cells. Furthermore, when in vitro cultured transgenic pre-B cells differentiated into immature B cells there was concomitant down-regulation of human CD122 and endogenous VpreB1. Thus the VpreB1 enhancer is sufficient to ensure developmental stage-specific expression of a reporter gene in B lymphocytes in vivo.

5' Flanking Region↗
PubMed1991

Close linkage of a transgene insertion site to the steel (Sl) locus on mouse chromosome 10.

The characterization of the insertion sites of exogenous sequences in transgenic mice can identify loci that are potentially useful for the genetic analysis of the mammalian genome. We have found that the transgene insertion site in the transgenic line TG.EB is tightly linked with the Steel (Sl) locus on mouse chromosome 10. In a backcross between doubly heterozygous transgenic Sl (Tg.EB +/+ Sl) mice and wild-type mice, only one recombinant was found in 135 progeny (recombination percentage = 0.7 +/- 0.7). The recombination frequency of the transgene with marker loci known to flank Sl was consistent with a map position close to Sl. Genomic sequences that are adjacent to the transgene insertion site were cloned and found to be tightly linked with the Sl locus in interspecific crosses using nontransgenic mice. Recombination analysis utilizing the transgene insertion site locus was used to show that a recently identified hematopoietic growth factor is encoded at Sl. The cloned sequences from the transgene insertion site are polymorphic in inbred strains of mice and can be utilized to determine the genotype at Sl during early embryonic development. Further, they may be useful in characterizing the genomic region near Sl that is affected in Sl deletion mutants.

Animals↗
PubMed1996

Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat.

The unique bread-making characteristic of wheat flour is closely related to the elasticity and extensibility of the gluten proteins stored in the starchy endosperm, particularly the high-molecular-weight glutenin subunits (HMW-GS), which are important in determining gluten and dough elasticity. The quality of wheat cultivars depends on the number and composition of the HMW-GS present. We have introduced the HMW-GS 1Ax1 gene, known to be associated with good bread-making quality, into the Bob White cultivar of wheat (Triticum aestivum L.), in which it is not present in nature, by the biolistic bombardment of cultured immature embryos. Of the 21 independent transformed lines selected, 20 expressed the selectable bar gene, and nine the 1Ax1 gene. The amount of HMW-GS 1Ax1 protein produced in the different transgenic lines varied from 0.6% to 2.3% of the total protein, resulting in an increase of up to 71% in total HMW-GS proteins. The transgenic plants were normal, fertile, and showed Mendelian segregation of the transgenes. The accumulation of HMW-GS 1Ax1 was consistent and stable up to the R3 seed generation. These results demonstrate that it is possible to manipulate both the quantity and quality of HMW-GS, which influence the bread-making quality of wheat.

Biotechnology↗