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At least 127 records · Page 7Linked to original sources
PubMed1997

Finite change analysis of glycolytic intermediates in tuber tissue of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase.

Genetically engineered organisms overexpressing phosphofructokinase (PFK), a supposed 'regulatory' step of glycolysis, often show little or no measurable change in glycolytic or respiratory flux, although the concentrations of glycolytic intermediates may change. We have used the finite change theory of Metabolic Control Analysis (MCA) to analyse the concentrations of glycolytic metabolites in aged disks of tuber tissue from four lines of transgenic potatoes expressing different amounts of PFK that, under aerobic conditions, showed statistically indistinguishable rates of respiration. The constancy of the metabolites' concentration deviation indices for different increases in PFK expression indicated that the metabolite changes from a graded series, excluding the possibility of anomalous behaviour that might be observed in a single transgenic line. Consequently we were able to use the finite change method to validate the results of an MCA model of tuber glycolysis [Thomas, Mooney, Burrell and Fell (1997) Biochem. J. 322, 119-127]. Furthermore the metabolite changes with PFK activity are evidence that near-equilibrium steps do not transmit increased substrate concentrations down the pathway without attenuation. Our results support the view that flux increase by activation of a single enzyme early in the pathway will, contrary to expectations, be of limited effectiveness in achieving flux increases.

Energy Metabolism↗
PubMed2006

HIV-1 genes vpr and nef synergistically damage podocytes, leading to glomerulosclerosis.

This study aimed to identify the causative gene for HIV-1 associated nephropathy, a paradigmatic podocytopathy. A previous study demonstrated that transgenic expression of nonstructural HIV-1 genes selectively in podocytes in mice with FVB/N genetic background resulted in podocyte injury and glomerulosclerosis. In this study, transgenic mice that expressed individual HIV-1 genes in podocytes were generated. Five of six transgenic mice that expressed vpr developed podocyte damage and glomerulosclerosis. Analysis of an established vpr transgenic line revealed that transgenic mice on FVB/N but not on C57BL/6 genetic background developed podocyte injury by 8 wk of age, with later glomerulosclerosis. Four of 11 transgenic mice that expressed nef also developed podocyte injury. One transgenic line was established from the nef founder mouse with the mildest phenotype. Transgenic mice in this line developed mesangial expansion at 3 wk of age and mild focal podocyte damage at 10 wk of age. Mating with FVB/N mice did not augment nephropathy. None of the transgenic mice that expressed vif, tat, rev, or vpu in podocytes, even with the FVB/N genetic background, developed podocyte injury. For testing effects of simultaneous expression of vpr and nef, these two lines were mated. All nef:vpr double-transgenic mice showed severe podocyte injury and glomerulosclerosis by 4 wk of age. In contrast, all vpr or nef single-transgenic mice in the same litter uniformly showed no or much milder podocyte injury. These findings indicate that vpr and nef each can induce podocyte injury with a prominent synergistic interaction.

AIDS-Associated Nephropathy↗
PubMed1998

Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus.

Tobacco plants were transformed with genes encoding the tobacco vein mottling potyvirus (TVMV) P1 or P3 protein. When compared with vector-transformed or P1 transgenic lines, seedlings of P3 transgenic lines (except a low expressor line) developed poorly, suggesting a detrimental effect of P3 on the plant. All P1 and P3 transgenic lines were protected against the homologous TVMV strain and showed variable proportions of two resistance phenotypes: asymptomatic plants or symptomatic plants that recovered from infection. The resistance was effective against a high inoculum dose but had a narrow spectrum. The heterologous strain TVMV-S was able to overcome resistance in most P1 lines but did not break the resistance of most P3 lines. No line showed resistance to another potyvirus (potato virus Y) or to potato virus X. These features and the low levels of transgene expression in resistant plants suggest that protection in P1 and P3 lines is RNA-mediated. In contrast with most reports on virus-activated gene silencing, some P3 lines with the predominant 'recovery' phenotype showed silencing of the transgene that was activated at a certain developmental stage of the plant independently of virus infection.

Gene Expression Regulation, Plant↗
PubMed2001

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.

Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.

Actins↗
PubMed1992

Effects of expression of human or bovine growth hormone genes on sperm production and male reproductive performance in four lines of transgenic mice.

Reproductive performance was studied in transgenic males from lines expressing and transmitting four hybrid genes: mouse metallothionein-I/human growth hormone (GH) (MT/hGH), MT/hGH placental variant (MT/hGH.V), MT/bovine GH (MT/bGH) and phosphoenolpyruvate carboxykinase/bGH (PEPCK/bGH). Each male was exposed to three normal females for 1 week and to three different normal females for another week. Females were examined for vaginal plugs and necropsied on day 14 of pregnancy. Males were killed for analysis of organ weights, numbers of testicular spermatids, numbers of epididymal sperm and measurements of plasma glucose concentration. Fertility of MT/hGH and MT/hGH.V transgenic males was significantly lower than in normal males, primarily because most males failed to impregnate any females. In females that became pregnant, the numbers of corpora lutea, total fetuses and live fetuses did not differ from those in females mated to normal (nontransgenic) males. Fetal crown-rump length on day 14 of pregnancy did not differ between litters sired by normal or by transgenic males. Weights of testes and seminal vesicles were significantly greater in all four types of transgenic male, but daily sperm production per unit weight (g-1) of testis was not affected and epididymal sperm reserves were either normal or slightly higher than normal. Plasma glucose concentrations were significantly higher in PEPCK/bGH mice than in other mice. Average or individual reproductive performance of transgenic males from the various lines did not correlate with any of the parameters examined except for significantly heavier seminal vesicles in MT/hGH and MT/hGH.V males than in normal males; these transgenic males exhibited a high incidence of infertility. Since hGH and hGH.V, but not bGH, are lactogenic in rodents, it was concluded that chronic stimulation of GH and prolactin receptors by ectopically produced human GHs in transgenic mice compromises male fertility by an unknown mechanism. Reduced fertility of transgenic males with MT/hGH or MT/hGH.V hybrid genes is due to failure to inseminate or impregnate females rather than to reduced numbers of spermatozoa or gross changes in the male reproductive system.

Animals↗
PubMed1989

Association of foreign DNA sequence with male sterility and translocation in a line of transgenic mice.

We have analyzed a line of transgenic mice derived from injection of a cloned human interferon cDNA. This line manifests total male sterility of males carrying the human sequence, while male littermates not harboring the foreign DNA are fertile. All females are fertile. Karyotypes of transgenic animals show 2:12 translocation. The microinjected sequence maps to one of the translocation chromosomes composed of a large portion of chromosome 12 to which has been translocated a segment of chromosome 2. Analysis of the sterile males reveals significant abnormalities of spermatogenesis and faulty chromosome synapsis that involves the translocation chromosomes. These findings show that transfer of foreign DNA into mouse embryos may lead to chromatin breakage and infertility of transgenic animals.

Animals↗
PubMed1995

A transgenic mouse line harboring a smooth muscle alpha-actin promoter polyomavirus middle T antigen transgene develops an epithelial hyperplasia in the rectum and distal stomach.

BACKGROUND: Transgenic mice are the product of the microinjection of foreign DNA directly into the pronuclei of a one-cell embryo. The foreign DNA can cause insertional inactivation or activation of the flanking genetic locus. EXPERIMENTAL DESIGN: We isolated five lines of transgenic mice harboring the chicken alpha-actin vascular smooth muscle enhancer/promoter linked to the polyomavirus middle T antigen using a standard microinjection protocol. The expression of the transgene was assessed in RNA prepared from affected and nonaffected tissue by RNase protection and reverse transcriptase-polymerase chain reaction analyses. Cell morphology was determined in stained sections from fixed tissues. RESULTS: In this article, we document the development of epithelial hyperplasia in the rectum and distal stomach together with female infertility in a single line of transgenic mice harboring the transgene. We were unable to demonstrate the expression of the transgene in any tissue examined, regardless of the degree of hyperplasia. The phenotype was present in the heterozygous state in both males and females. CONCLUSIONS: In the absence of the expression of the transgene, we conclude that the insertion of the transgene may have caused the epithelial hyperplasia directly or may have contributed to a condition that promotes hyperplasia. The transgene may have activated a dominant-acting neighboring gene.

Actins↗
PubMed2004

Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice.

Stem borer resistant transgenic parental lines, involved in hybrid rice, were produced by Agrobacterium-mediated gene transfer method. Two pSB111 super-binary vectors containing modified cry1Ab/cry1Ac genes driven by maize ubiquitin promoter, and herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter were, used in this study. Embryogenic calli after co-cultivation with Agrobacterium were selected on the medium containing phosphinothricin. Southern blot analyses of primary transformants revealed the stable integration of bar, cry1Ab and cry1Ac coding sequences into the genomes of three parental lines with a predominant single copy integration and without any rearrangement of T-DNA. T1 progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. Furthermore, the co-segregation of bar and cry genes in T1 progenies suggested that the transgenes are integrated at a single site in the rice genome. In different primary transformants with alien inbuilt resistance, the levels of cry proteins varied between 0.03 and 0.13% of total soluble proteins. These transgenic lines expressing insecticidal proteins afforded substantial resistance against stem borers. This is the first report of its kind dealing with the introduction of Bacillus thuringiensis (Bt) cry genes into the elite parental lines involved in the development of hybrid rice.

Animals↗
PubMed2002

Tonin and kallikrein in the brain of transgenic rat line expressing human tissue kallikrein.

A transgenic rat line harboring the human tissue kallikrein gene was investigated for expression and activity of tonin and kallikrein in different regions of the brain. The introduction of the transgene into the rat genome produced a significant augmentation of the expression levels and activity of rat tissue kallikrein. The possibility that human kallikrein does not hydrolyze the rat substrate is probably responsible for the augmented expression of the rat enzyme. On the other hand, although expression of tonin was significantly reduced, tonin activity was not altered in most brain structures, except for cerebellum and neurohypophysis.

Animals↗
PubMed2006

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

Agrobacterium tumefaciens↗
PubMed2005

Transgenic mouse lines expressing synaptopHluorin in hippocampus and cerebellar cortex.

We generated six transgenic mouse lines in which synaptopHluorin (SpH), one of green fluorescent protein-based sensors of vesicular exocytosis, was expressed under the control of neuron-specific Thy-1.2 promoter. In situ hybridization study revealed that SpH mRNA was expressed in a broad spectrum of brain regions in four of them, whereas in others it was expressed in the specific regions of the hippocampus. In one particular line, SpH immunoreactivity was specifically observed in the mossy fiber presynaptic terminals of both hippocampus and cerebellar cortex. The fluorescence intensity of these presynaptic terminals was somewhat decreased by acidic buffer superfusion and greatly increased by vesicular neutralization of pH, indicating that the SpH molecules are mainly distributed in the synaptic vesicles. The exocytosis-dependent fluorescence increment was measured upon activation of a single presynaptic terminal. These transgenic lines are expected to facilitate morphological and physiological studies of presynaptic terminals in a variety of regions of the brain.

Animals↗
PubMed2005

Reestablishment of a transgenic rabbit line by artificial insemination using cryopreserved semen.

The production of recombinant proteins in the milk of transgenic animals is an alternative to traditional cell culture methodology. Transgenic rabbits can serve in the small-scale production of recombinant proteins, underscoring the need to maintain valuable transgenic lines. In this study, the authors used cryopreserved transgenic rabbit semen to artificially inseminate does, demonstrating the utility of this method for the reestablishment of a transgenic rabbit herd.

Animal Husbandry↗
PubMed1995

Differential susceptibility to tolerance induction in vitro of splenic B cells from several transgenic mouse lines. Role of B1 cells.

Spleen cells from transgenic mice, whose rearranged Ig receptors reflect the repertoires of B1 (CD5+ and "sister" B cells) or normal B2 cells, were examined for their ability to be rendered unresponsive. By using an anti-Ig tolerance protocol that is independent of receptor specificity, we previously reported that peritoneal B cells, containing primarily CD5+ and "sister" B cells, were not susceptible to unresponsiveness. Herein, we show that splenic B cells from two separate transgenic mouse lines, each expressing a B1-type receptor, are resistant to tolerance induction in vitro. In contrast, splenic B cells from two other transgenic mouse lines with a large representation of conventional B cells were sensitive to anti-lg-mediated unresponsiveness. This difference does not reside in the surface Ig density, cell cycle, or activation stage of these cells, but is reflected in the initial calcium mobilization and tyrosine phosphorylation after surface Ig cross-linking. Therefore, these results support the hypothesis that the antigenic specificity of B cell receptors may drive cells toward the B1 subset, as suggested by Cong et al. (Cong, Y-Z., E. Rabin, and H. H. Wortis. 1991. Int. Immunol. 3:467-476), and that B1 cell characteristics confer the ability of B cells to withstand in vitro tolerance induction, irrespective of their anatomical location. The possibility that this results from previous antigenic experience is discussed.

Animals↗
PubMed2000

Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

Animals↗
PubMed2004

Analysis of distinct tartrate-resistant acid phosphatase promoter regions in transgenic mice.

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Acid Phosphatase↗
PubMed1991

Characterization of a transgenic mouse line over-expressing the human ornithine decarboxylase gene.

We have produced several transgenic mouse lines over-expressing the human ornithine decarboxylase (ODC) gene. We have now characterized one of the transgenic lines as regards the tissue accumulation of the polyamines and the activities of their metabolizing enzymes. Among the tissues analysed, the polyamine pattern was most strikingly changed in testis and brain of the transgenic animals. ODC activity was greatly enhanced in all tissues, except kidney, of the transgenic animals. The most dramatic increase, 80-fold, was found in brain of the transgenic mice. The activities of S-adenosylmethionine decarboxylase and spermidine and spermine syntheses were likewise significantly increased in testis of the transgenic animals. The activities of the enzymes involved in the back-conversion of the polyamines, namely spermidine/spermine acetyltransferase and polyamine oxidase, were similar in the transgenic and non-transgenic animals. As analysed by reverse transcriptase/polymerase chain reaction, all the six tissues of the transgenic animals expressed human-specific ODC mRNA. Determination of the half-life of testicular ODC revealed a stabilization of the enzyme in the transgenic males.

Animals↗
PubMed2004

Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal.

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.

Fungi↗
PubMed2001

Transgenic tobacco plants expressing the maize Cat2 gene have altered catalase levels that affect plant-pathogen interactions and resistance to oxidative stress.

Transgenic tobacco genotypes expressing the maize Cat2 gene were developed with altered catalase (CAT) levels that resulted in a moderate increase of CAT activity in two transgenic lines. Bacterial infection, with a pathogen that does not share homology with the transgene, caused local and systemic down-regulation of the steady state mRNA levels of the 35S-driven transgene in a manner resembling post-transcriptional gene silencing (PTGS). Phenotypic symptoms of hypersensitive response (HR) and systemic acquired resistance (SAR) were similar in control SR1 and the transgenic genotypes. Induction of hin1, used as a molecular marker of plant responses to invading bacteria, displayed a similar pattern between control and transgenic lines, but some variation in the levels of expression was observed. The major difference was recorded in the ability of the plants to restrict bacterial growth during HR. All transgenic lines were more sensitive than control SR1, with two lines exhibiting a significantly reduced capacity to inhibit bacterial growth. This is consistent with the putative enhanced capacity of transgenic lines containing the maize Cat2 gene to more effectively remove H2O2, which may act as a direct antimicrobial agent. Steady state mRNA levels of PR-1 and PR-5 varied among the genotypes, possibly indicating differences in strength of the SAR signal. Transgenic line 2, which was the most sensitive during HR, was most effective in restricting bacterial growth during SAR. This indicates that a reverse correlation might exist between the severity of infection during HR and the ability to inhibit bacterial growth during SAR. Growth under high light conditions affected plant-pathogen interactions in control SR1, as well as in transgenic line 8. Early induction and higher expression of PR-1 and PR-5 was detected in both SR1 and line 8 in high light-grown plants as compared with their low light-grown counterparts. Our data indicate that growth under high light conditions can predispose plants to better resist pathogen attack, and may amplify local and systemic defense signals. Finally, one transgenic line, which exhibited 1.3-fold higher average CAT activity in comparison with the untransformed SR1 control, suffered significantly less methyl viologen (MV) damage than untransformed control plants at moderate and high MV concentrations.

Blotting, Southern↗