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At least 793 records · Page 44Linked to original sources
PubMed1999

Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model of familial amyotrophic lateral sclerosis.

A subset of familial cases of amyotrophic lateral sclerosis are linked to missense mutations in copper/zinc superoxide dismutase type 1. Patients with missense mutations in copper/zinc superoxide dismutase type 1 develop a paralytic disease indistinguishable from sporadic amyotrophic lateral sclerosis through an unknown toxic gain of function. Nitric oxide reacts with the superoxide anion to form the strong oxidant, peroxynitrite, which participates in neuronal injury in a variety of model systems. Peroxynitrite is an alternate substrate for copper/zinc superoxide dismutase type 1, causing catalytic nitration of tyrosine residues in other proteins. Mutations in copper/zinc superoxide dismutase type 1 may disrupt the active site of the enzyme and permit greater access of peroxynitrite to copper, leading to increased nitration by peroxynitrite of critical cellular targets. To investigate whether neuronal-derived nitric oxide plays a role in the pathogenesis of familial amyotrophic lateral sclerosis, we examined the effects of three different nitric oxide synthase inhibitors: a non-selective nitric oxide synthase inhibitor, nitro-L-arginine methyl ester; a relatively selective inhibitor of neuronal nitric oxide synthase, 7-nitroindazole; and a novel highly selective neuronal nitric oxide synthase inhibitor, AR-R 17,477, in transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 (Gly-->Ala at position 93; G93A) containing a high transgene copy number and a low transgene copy number. AR-R 17,477, but not nitro-L-arginine methyl ester or 7-nitroindazole, significantly prolonged survival in both the high and low transgene transgenic mice. To determine whether neuronal nitric oxide synthase is involved in the pathogenesis resulting from the familial amyotrophic lateral sclerosis copper/zinc superoxide dismutase type 1 mutation, we produced mice with the copper/zinc superoxide dismutase type 1 mutation which lack the neuronal nitric oxide synthase gene. The transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background do not live significantly longer than transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1. Western blot analysis indicates the presence of two neuronal nitric oxide synthase-like immunoreactive bands in spinal cord homogenates of the neuronal nitric oxide synthase null mice, and residual neuronal nitric oxide synthase catalytic activity ( > 7%) is detected in the spinal cord of the transgenic mice expressing a familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 on neuronal nitric oxide synthase null background. This amount of residual activity probably does not account for lack of protection afforded by the disrupted neuronal nitric oxide synthase gene in the familial amyotrophic lateral sclerosis-linked mutant human copper/zinc superoxide dismutase type 1 mice. Immunological nitric oxide synthase is not detected in the copper/zinc superoxide dismutase type 1 mutant mice at several different ages, thus excluding immunological nitric oxide synthase as a contributor to the pathogenesis of familial amyotrophic lateral sclerosis. Levels of neuronal nitric oxide synthase as well as Ca2+-dependent nitric oxide synthase catalytic activity in the copper/zinc superoxide dismutase type 1 mutant mice do not differ from wild type mice. Endothelial nitric oxide synthase levels may be decreased in the copper/zinc superoxide dismutase type 1 mutant mice. Together, these results do not support a significant role for neuronal-derived nitric oxide in the pathogenesis of familial amyotrophic lateral sclerosis transgenic mice.

Amidines↗
PubMed2000

Upregulation of an opioid-mediated antinociceptive mechanism in transgenic mice over-expressing substance P in the spinal cord.

In transgenic mice expressing ectopic substance P fibres in the spinal white matter, a normally innocuous mechanical stimulus induces hyperalgesia and allodynia which are reversed by substance P and N-methyl-D-aspartate receptor antagonists. This period of enhanced excitation is followed by a rebound overshoot in these animals. As previous evidence indicates opioid mechanisms in a similar rebound in normal animals, the present study was done to determine the effects of systemic administration of morphine and the opiate receptor antagonist, naloxone, on the stimulus-induced responses in the tail withdrawal reflex. Once baseline reaction times had been taken, different combinations of saline, naloxone and morphine were administered intraperitoneally to transgenic and control mice of either sex. A mechanical conditioning stimulus of 450g was then applied to the tip of the tail for 2s. This stimulus was innocuous in control mice given saline or naloxone, but provoked a nociceptive response in transgenic mice given these compounds. In control and transgenic mice, following morphine administration there was an antinociceptive effect. In control mice the subsequent mechanical stimulus had no effect. However, in transgenic mice the mechanical stimulus produced a further antinociception. Naloxone blocked the effect of morphine and the subsequent conditioning stimulus in both control and transgenic mice. The results indicate that while morphine is equally effective on the withdrawal reflex in both types of animal, in the transgenic mice morphine reveals an intrinsic, naloxone-sensitive antinociceptive mechanism. The data are interpreted to suggest that over-expression of substance P or some other factor in the spinal cord of transgenic mice is associated with the up-regulation or facilitation of an opiate-mediated intrinsic antinociceptive mechanism. This is a novel observation because the genetic manipulation in this transgenic mouse results in a transient over-expression of nerve growth factor during development that leads to the formation of ectopic primary afferent fibres in the spinal cord containing substance P. These fibres persist indefinitely after the nerve growth factor levels return to normal. Opioid mechanisms, which are likely of dorsal horn origin, do not fall under the direct influence of nerve growth factor mechanisms and therefore the intriguing possibility is raised that opioid mechanisms in the spinal cord are regulated at least in part by substance P-related mechanisms.

Analgesics, Opioid↗
PubMed2000

Transgenic livestock: premises and promises.

Microinjection of DNA constructs into pronuclei of zygotes has been the method of choice for the generation of transgenic livestock. However, this procedure is characterized by low efficiency (1-4% transgenic offspring), random integration and variable expression of the transgene as well as a considerable proportion of mosaicism. Furthermore, it is extremely time consuming and costly. As a consequence, commercial application has focused on the production of recombinant proteins in the mammary gland of transgenic animals and xenotransplantation, e.g. the use of porcine organs in human organ transplantation. In addition, transgenic pigs carrying a modified porcine growth hormone (hMt-pGH) construct show significant improvements in economically important traits without adverse side effects of a GH overproduction. Improvements of transgenic technology will likely come from the generation of appropriate cell lines suitable for transfection or even homologous recombination and their subsequent use in nuclear transfer. Additionally, in the mouse a number of sophisticated molecular tools have been developed that allow precise modifications of the genome. These include the application of artificial chromosomes from yeast (YAC) or bacteria (BAC) for position-independent and copy-number-dependent expression of a transgene, the Tet-system (tetracycline inducible) for a tight temporal control of transgene expression, as well as conditional mutagenesis by applying site-specific DNA recombinases (e.g. Cre, FLP). The successful adaptation of these molecular tools to livestock will enable the fulfillment of many of the promises originally thought to be achievable when transgenic livestock were first reported.

Animals↗
PubMed2002

Prosaposin: threshold rescue and analysis of the "neuritogenic" region in transgenic mice.

Prosaposin is the precursor of four glycoprotein activators (saposins) for lysosomal hydrolases. Intact prosaposin also has lipid transfer properties in vitro as well as neuritogenic effects ex vivo and in vivo. Such "neuritogenic" effects of saposin C were evaluated in vivo using transgenic mice with prosaposin cDNAs having normal (PS-N) or mutated neuritogenic region. The mutant prosaposin cDNA (PS-CBC) encoded a chimeric saposin C that contained the non-neuritogenic sequence of saposin B, but retained acid beta-glucosidase (GCase) activation effects. When driven by the PGK (3-phosphoglycerate kinase) promoter, transgene expression was highest in the cerebrum for any of the transgenes (range from 15% to 42% of wild-type). Low levels were in visceral tissues. Prosaposin knock-out (PS-/-) mice expressing N or CBC transgenes, even at low levels, had delayed onset of neurologic signs and neuropathology, and significant lengthening of life span (from 1.7- to 7-fold) with age dependent partial correction of GlcCer and LacCer accumulation in the brain. Neuropathologic progression and neuronal glycosphingolipid storage were related directly to the transgene expression levels in the brain. Purkinje cell loss was age dependent. Gross brain and neuronal organizations were indistinguishable in PS-/- mice with or without the various transgenes, albeit the phenotype appeared later in the mice with transgenes. These studies show the degree of neuropathologic manifestations in each transgenic line depended on expression level rather than on the nature of the transgene. These studies also show in vivo localization of the GCase activation region to the carboxy terminal half of saposin C and the lack of a significant gross trophic effect of saposin C on CNS organization in vivo.

Animals↗
PubMed2001

Abnormal ornithine decarboxylase activity in transgenic mice increases tumor formation and infertility.

A transgenic mouse line carrying ornithine decarboxylase cDNA as the transgene under the control of a mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter was generated in order to study whether ornithine decarboxylase transgene expression will have any physiological or pathological effect during the entire life of a transgenic mouse. The high frequency of infertile animals and the loss of pups made the breeding of homozygous mice unsuccessful. However, a colony of heterozygous transgenic mice was followed for 2 years. In adult heterozygous transgenic mice, ornithine decarboxylase activity was significantly increased in the testis, seminal vesicle and preputial gland when compared to non-transgenic controls. In contrast, ornithine decarboxylase activity was decreased in the kidney and prostate of transgenic mice. No significant changes in ornithine decarboxylase activity were found in the ovary and mammary gland and only moderate changes in ornithine decarboxylase activity were detected in the heart, brain, pancreas and lung. The most common abnormalities found in adult animals (12 males and 20 females) of the transgenic line were inflammatory processes, including pancreatitis, hepatitis, sialoadenitis and pyelonephritis. Spontaneous tumors were observed in eight animals, including two benign tumors (one dermatofibroma, one liver hemangioma) and six malignant tumors (one lymphoma, one intestinal and three mammary adenocarcinomas and one adenocarcinoma in the lung). No significant pathological changes were found in 17 nontransgenic controls.

Animals↗
PubMed2000

Transgenic farm animals: applications in agriculture and biomedicine.

During the last decade, tremendous progress has been made in the area of transgenic farm animals. While there are many important transgenic farm animal applications in agriculture, funding has been very limited and progress has been rather slow in this area. Encouragingly, the potential applications of transgenic farm animals as bioreactors for producing human therapeutic proteins and as organ donors for transplantations in humans have attracted vast funding from the private sectors. Several transgenic animal products are already in various phases of clinical trials. Estimates are, that in the near future, the worlds demands on human pharmaceutical proteins may largely be met by transgenic farm animals. While there are still major challenges ahead in the area of xenotransplantation using transgenic animal organs, transgenic tissues or cells have demonstrated promising results as a potential tool for gene therapy. Recent development on cloning, embryonic stem cells and alternative transgenic methods may further expand the transgenic applications in both agriculture and biomedicine.

Agriculture↗
PubMed1998

High-level constitutive expression of alpha 1-acid glycoprotein and lack of protection against tumor necrosis factor-induced lethal shock in transgenic mice.

alpha 1-Acid glycoprotein (AGP) is an acute phase protein produced by hepatocytes. Although its exact biological function remains controversial, it was shown to protect galactosamine-sensitized or normal mice against hepatitis and lethal shock induced by tumor necrosis factor (TNF). Rat-AGP-transgenic mice, constitutively producing several mg AGP per ml serum were tested for their response to a combined challenge with TNF and D-(+)-galactosamine. A previously characterized, single transgenic line (9.5-5) was used. In contrast to our expectations both heterozygous or homozygous transgenic mice were not protected by the endogenously overproduced AGP. However, both transgenic and non-transgenic mice were protected by pretreatment with interleukin-1, an effect which we believe is mediated by the induction of acute phase proteins like AGP. Furthermore, both types of mice were protected by exogenous bovine AGP, suggesting that the lack of protection by endogenous AGP is not because of a repressed response to AGP. Finally, we demonstrate that purified AGP from the serum of transgenic mice is as protective as the AGP from non-transgenic mice or rats. The results suggest that AGP is protective only when its concentration is rapidly induced, perhaps because the endogenous steady state synthesis of AGP, in non-transgenic as well as transgenic mice, is coupled to the production of an AGP-binding factor. This study provides an interesting example of differences in outcome to a lethal challenge between an acute administered and a chronically produced protective protein.

Acute-Phase Reaction↗
PubMed1998

Overexpression of growth hormone affects alternatively spliced IGF-I mRNA expression in oMt1a-oGH transgenic mice.

Restorative growth hormone (GH) treatment of hypophysectomized rats differentially enhances the transcription of alternative IGF-I mRNA classes in liver. The goal of the present study was to determine the effects of GH overexpression on various classes of hepatic IGF-I mRNA in GH transgenic mice. Unstimulated oMt1a-oGH transgenic mice had low levels of transgene expression, and therefore were used to determine the effects of long-term, slightly elevated GH levels on the abundance on each alternative IGF-I mRNA class. The acute effects of high GH levels on the expression of alternative IGF-I mRNA were studied by gavaging transgenic mice with 25 mM zinc sulfate to activate oMt1a-oGH transgene expression. Long-term, low levels of oGH transgene expression in unstimulated transgenic mice resulted in a 73% down regulation of IGF-I 2Ea mRNA but not 1Ea and 2Eb mRNA. Acute stimulation of transgene expression triggered a rapid, 240% increase in 1Ea mRNA levels within 4 hours of transgene expression while 2Ea mRNA was down regulated to nearly non-detectable levels by 6 hours. IGF-I 2Eb mRNA was not affected by the short-term GH elevation. Our results showed that IGF-I 1Ea and 2Ea mRNA were differentially regulated by chronic low or acute high levels of GH. These results suggest that the regulation of IGF-I 1Ea and 2Ea mRNA transcription involve different postreceptor molecules and/or feedback mechanisms.

Alternative Splicing↗
PubMed1997

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice.

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken 2) genetic background. This effect was not seen with zmZ27 or osGTI transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm.

Blotting, Southern↗
PubMed1997

Actions and interactions of growth hormone and insulin-like growth factor-II: body and organ growth of transgenic mice.

To characterize long-term actions and interactions of growth hormone (GH) and insulin-like growth factor-II (IGF-II) on postnatal body and organ growth, hemizygous phosphoenolpyruvate carboxykinase (PEPCK)-human IGF-II transgenic mice were crossed with hemizygous PEPCK-bovine GH transgenic mice. The latter are characterized by two-fold increased serum levels of IGF-I and exhibit markedly increased body, skeletal and organ growth. Four different genetic groups were obtained: mice harbouring the IGF-II transgene (I), the bGH transgene (B), or both transgenes (IB), and non-transgenic controls (C). These groups of mice have previously been studied for circulating IGF-I levels (Wolf et al., 1995a), whereas the present study deals with body and organ growth. Growth curves (week 3 to 12) were estimated by regression with linear and quadratic components of age on body weight and exhibited significantly (p < 0.001) greater linear coefficients in B and IB than in I and C mice. The linear coefficients of male I and C mice were significantly (p < 0.001) greater than those of their female counterparts, whereas this sex-related difference was absent in the bGH transgenic groups. The weights of internal organs as well as the weights of abdominal fat, skin and carcass were recorded from 3.5- to 8-month-old mice. In addition, organ weight-to-body weight-ratios (relative organ weights) were calculated. Except for the weight of abdominal fat, absolute organ weights were as a rule significantly greater in B and IB than in I and C mice. IGF-II overproduction as a tendency increased the weights of kidneys, adrenal glands, pancreas and uterus both in the absence and presence of the bGH transgene. Analysis of relative organ weights demonstrated significant (p < 0.05) effects of elevated IGF-II on the relative growth of kidneys (males and females) and adrenal glands (females), confirming our previous report on organ growth of PEPCK-IGF-II transgenic mice. In females, IGF-II and GH overproduction were additive in stimulating the growth of spleen and uterus, providing evidence for tissue-specific postnatal growth promoting effects by IGF-II in the presence of elevated IGF-I.

Abdomen↗
PubMed2003

Strain-dependent differences in the efficiency of transgenic mouse production.

Transgenic mouse production via pronuclear microinjection is a complex process consisting of a number of sequential steps. Many different factors contribute to the effectiveness of each step and thus influence the overall efficiency of transgenic mouse production. The response of egg donor females to superovulation, the fertilization rate, egg survival after injection, ability of manipulated embryos to implant and develop to term, and concentration and purity of the injected DNA all contribute to transgenic production efficiency. We evaluated and compared the efficiency of transgenic mouse production using four different egg donor mouse strains: B6D2/F1 hybrids, Swiss Webster (SW) outbred, and inbred FVB/N and C57BL/6. The data included experiments involving approximately 350 DNA transgene constructs performed by a high capacity core transgenic mouse facility. Significant influences of particular genetic backgrounds on the efficiency of different steps of the production process were found. Except for egg production, FVB/N mice consistently produced the highest efficiency of transgenic mouse production at each step of the process. B6D2/F2 hybrid eggs are also quite efficient, but lyze more frequently than FVB/N eggs after DNA microinjection. SW eggs on the other hand block at the 1-cell stage more often than eggs from the other strains. Finally, using C57BL/6 eggs the main limiting factor is that the fetuses derived from injected eggs do not develop to term as often as the other strains. Based on our studies, the procedure for transgenic mouse production can be modified for each egg donor strain in order to overcome any deficiencies, and thus to increase the overall efficiency of transgenic mouse production.

Animals↗
PubMed2003

Methylation of the exon/intron region in the Ubi1 promoter complex correlates with transgene silencing in barley.

Methylation of plant promoters is often associated with transcriptional gene silencing, while methylation of the transcribed region is associated with post-transcriptional gene silencing. Promoter complexes that include the first untranslated exon and intron, such as maize ubiquitin1 and rice actin1, have been widely used in monocot transformation because they support higher levels of transient transgene expression than the core promoter does. However, persistent problems with transgene silencing driven by these promoter complexes brought up a troubling question: were higher initial levels of transgene expression at the expense of long-term expression stability? Here we report that methylation of an untranslated exon and intron in the promoter complex is correlated with transcriptional transgene silencing in barley. Two sibling sublines, designated T(3)30 and T(3)31, were identified in a homozygous T3 population from a single transgenic parental line. In the T6 generation, all progeny of one subline, T(3)30, expressed ubiquitin-driven bar and uidA, but both transgenes were transcriptionally silenced in the other subline, T(3)31. Although the structure of the transgene locus is complex, no differences in the physical structure or location of the locus were detected between the two sublines. Transcriptional transgene silencing in T(3)31 correlated with two molecular events: methylation of the first untranslated exon and 5' end of the intron of the promoter complex, and condensation of the chromatin in regions containing transgenes. Passage of the non-silenced subline through in vitro culture recreated the silenced phenotype of T(3)31 and the molecular events leading to its silencing.

5' Flanking Region↗
PubMed2002

Evaluation of Tet-on system to avoid transgene down-regulation in ex vivo gene transfer to the CNS.

Ex vivo gene transfer to the CNS has so far been hampered by instability of transgene expression. To avoid the phenomenon of transgene down-regulation, we have employed strong, constitutive promoters and compared this expression system with the inducible Tet expression system incorporated in a single plasmid vector or in lentiviral vectors. Plasmid-based transgene expression directed by the constitutive, human ubiquitin promoter, UbC, was stable in transfected HiB5 cells in vitro and comparable in strength to the CMV promoter. However, after transplantation of UbC and CMV HiB5 clones to the rat striatum, silencing of the transgene occurred in most cells soon after implantation of transfected cells. The Tet-on elements were incorporated in a single plasmid vector and inducible HiB5 clones were generated. Inducible clones displayed varying basal expression activity, which could not be ascribed to an effect of cis-elements in the vector, but rather was due, at least in part, to intrinsic activity of the minimal promoter. Basal expression activity could be blocked in a majority of cells by stable expressing the transrepressor tTS. Fully induced expression levels were comparable to CMV and UbC promoters. Similar to the constitutive promoters transgene expression was down-regulated soon after grafting of inducible HiB5 clones to the rat striatum. Lentiviral vectors can direct long-term stable in vivo transgene expression. To take advantage of this quality of the lentiviral vector, the Tet-on elements were incorporated in two lentiviral transfer vectors followed by transduction of Hib5 cells. Interestingly, all HiB5 clones established by lentiviral transduction showed very similar expression patterns and tight regulatability that apparently was independent of transgene copy number and integration site. Nevertheless, transgene expression in all lentiviral HiB5 clones was down-regulated shortly after transplantation to the rat striatum. These results confirm the general phenomenon of transgene down-regulation. Moreover, the results suggest that the considerable advantages offered by lentiviral vectors for direct gene delivery cannot necessarily be transferred directly to ex vivo gene delivery. This emphasizes the need for alternative vector strategies for ex vivo gene transfer.

Animals↗
PubMed2003

Increased fatty acid oxidation in transgenic mice overexpressing UCP3 in skeletal muscle.

AIM: To determine the rates of substrate oxidation by skeletal muscle in vitro as well as tissue-specific glucose uptake in vivo in transgenic mice overexpressing uncoupling protein-3 (UCP3) in skeletal muscle. METHODS: Soleus muscle was isolated from transgenic mice overexpressing UCP3 in skeletal muscle and wild-type mice. Rates of [1-14C]-palmitate oxidation and [2-14C]-pyruvate oxidation were determined by in vitro incubation of the soleus muscle. Tissue glucose uptake rates were characterized during a glucose tolerance test using 2-deoxy-[1-3H]-glucose as a tracer. RESULTS: Oxidation of [1-14C]-palmitate to CO2 by isolated soleus muscle was increased in UCP3 transgenic mice (0.45 +/- 0.03 vs. 0.24 +/- 0.02 micro mol/h/g). [2-14C]-pyruvate oxidation, which is a measure of the activity of pyruvate carboxylase in introducing pyruvate carbon into the tricarboxylic acid cycle, was increased 1.4-fold in the presence of fatty acid in the UCP3 transgenic mice (3.84 +/- 0.28 vs. 5.36 +/- 0.29 micro mol/h/g). The plasma glucose concentration after an overnight fast was significantly lower in the UCP3 transgenic mice (3.56 +/- 0.37 vs. 5.11 +/- 0.33 m/mol). Only brown adipose tissue from the UCP3 transgenic mice showed increased tissue glucose uptake rates compared with the wild-type mice. Skeletal muscle uptake rates of 2-deoxyglucose were either unchanged (soleus and gastrocnemius) or reduced (diaphragm) in the UCP3 transgenic mice. CONCLUSIONS: The improved glucose tolerance in the UCP3 transgenic mice does not appear to be the result of increased uptake into peripheral tissues. The increased fatty acid oxidation in skeletal muscle of UCP3 transgenic mice supports the proposed role of UCP3 in the export of fatty acid anions from mitochondria during fatty acid oxidation.

Animals↗
PubMed1999

Inducible nitric oxide synthase up-regulation in a transgenic mouse model of familial amyotrophic lateral sclerosis.

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.

Alanine↗
PubMed2001

Relation of skin polyamines to the hairless phenotype in transgenic mice overexpressing spermidine/spermine N-acetyltransferase.

We recently generated a transgenic mouse line with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase. Phenotypic changes in these animals included permanent loss of hair at the age of 3 wk. We have now further explored development of hair loss during early postnatal life. The first hair cycle appeared to be completed normally in the transgenic animals. At postnatal day 15, although macroscopically indistinguishable from their syngenic littermates, the transgenic animals already showed microscopically signs of hair follicle degeneration. Wild-type mice started their second anagen phase at day 27, whereas the transgenic animals did not display functional hair follicles at that time. Hair follicles were replaced by dermal cysts and epidermal utriculi. Analysis of skin polyamines revealed that the transgenic animals continuously overaccumulated putrescine. The view that an overaccumulation of putrescine was related to the disturbed hair follicle development was strengthened by the finding that doubly transgenic mice overexpressing, both spermidine/spermine N1-acetyltransferase and ornithine decarboxylase and with extremely high levels of putrescine in the skin, showed distinctly more severe skin changes compared with the singly transgenic animals. Interest ingly, in spite of their hairless phenotype, the spermidine/spermine N1-acetyltransferase transgenic mice, were significantly more resistant to the development of papillomas in response to the two-stage skin carcinogenesis. Analysis of skin polyamines indicated that the syngenic mice tripled their spermidine content when exposed to promotion, whereas the transgenic animals showed only modest changes. These results suggest that putrescine plays a pivotal part in normal hair follicle development.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice.

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

Animals↗
PubMed1991

Heterochromatic features of an 11-megabase transgene in brain cells.

Transgenic mice provide a remarkable experimental setting for the study of nuclear architecture. The three-dimensional localization and fine structure of a foreign DNA within the mouse genome can be conveniently followed by using high-resolution in situ hybridization. Foreign DNAs designed with specific characteristics, such as base bias, sequence motif(s), and size can stably integrate into finite positions on host chromosomes. Thus the relative importance of each of these characteristics in determining the three-dimensional nuclear position and the detailed morphology of the transgene can be evaluated in different cell types. The aim of this study was to evaluate a transgene with sequence characteristics that might contribute to the de novo formation of heterochromatin in interphase nuclei. The structure of a phenotypically silent 11-megabase transgene, containing tandem repeats of beta-globin-pBR sequences integrated into the peritelomeric region of both mouse chromosome 3 homologs, was determined in adult brain cells. Neurons that are largely euchromatic were especially informative in three-dimensional studies of transgene position. The two transgenic loci behaved much like centromeric or paracentromeric A + T-rich satellite DNAs of comparable length from a single chromosome; one or both transgene domains localized together with centromeric satellite DNA on the nucleolus. This is an unusual nuclear position for a telomeric or chromosome arm region that does not contain a substantial amount of constitutively heterochromatic satellite DNA. G + C richness did not prevent these regions from assembling as dense heterochromatic bodies of approximately 1 micron3 in volume. Ultrastructurally, transgenic domains were often intimately connected with constitutive heterochromatin and were highly condensed. Labeled supercoils, formed by a discrete approximately 250-nm-wide fiber, were observed in oblique thin sections through the center of the domain. The structural data were consistent with negligible transcriptional activity detected for this locus, as well as the predicted conformation of constitutive heterochromatin. Interestingly, in transgenic but not control mice, a substantial number of large neurons, including approximately 30% of cerebellar Purkinje cells, showed excessive invaginations of the nuclear membrane.

Animals↗