scholarly journals Transgenic mice over-expressing the human spermidine synthase gene

1993 ◽  
Vol 293 (2) ◽  
pp. 513-516 ◽  
Author(s):  
L Kauppinen ◽  
S Myöhänen ◽  
M Halmekytö ◽  
L Alhonen ◽  
J Jänne
Keyword(s):  
Transgenic Mice ◽  
Reverse Transcriptase ◽  
Human Gene ◽  
Transgenic Animals ◽  
Chromosome 1 ◽  
Pcr Analysis ◽  
Spermidine Synthase ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Adenosylmethionine Decarboxylase

We have generated a transgenic mouse line harbouring the functional (chromosome-1-derived) human spermidine synthase (EC 2.5.1.16) gene in their genome. The transgenic animals expressed the human gene-derived mRNA, as revealed by reverse-transcriptase/PCR analysis, in all tissues studied and displayed tissue spermidine synthase activity that was 2-6 times that in their syngenic littermates. The elevated spermidine synthase activity, however, had virtually no effect on tissue putrescine, spermidine or spermine levels. The view that the accumulation of spermidine and spermine is possibly controlled by S-adenosylmethionine decarboxylase was further supported by the finding that tissue spermidine and spermine contents also remained practically normal in hybrid transgenic mice over-expressing both human ornithine decarboxylase and spermidine synthase genes.

scholarly journals Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue

1999 ◽  
Vol 338 (2) ◽  
pp. 311-316 ◽  
Author(s):  
Suvikki SUPPOLA ◽  
Marko PIETILÄ ◽  
Jyrki J. PARKKINEN ◽  
Veli-Pekka KORHONEN ◽  
Leena ALHONEN ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Cell Injury ◽  
Mrna Level ◽  
Transgenic Animals ◽  
Ultrastructural Changes ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Polyamine Analogue

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT–SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and > 90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1–2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.

scholarly journals Transgenic mice over-producing putrescine in their tissues do not convert the diamine into higher polyamines

1993 ◽  
Vol 291 (2) ◽  
pp. 505-508 ◽  
Author(s):  
M Halmekytö ◽  
L Alhonen ◽  
L Alakuijala ◽  
J Jänne
Keyword(s):  
Transgenic Mice ◽  
Transgenic Mouse ◽  
Ornithine Decarboxylase ◽  
Specific Inhibitor ◽  
Mouse Line ◽  
Transgenic Mouse Line ◽  
Adenosylmethionine Decarboxylase ◽  
Adult Tissues ◽  
Ornithine Transaminase

We recently described a transgenic mouse line over-expressing the human ornithine decarboxylase gene virtually in all tissues. Despite strikingly elevated tissue putrescine concentrations, no or minimal changes were found in the levels of the higher polyamines spermidine and spermine. We have now extended these studies by further increasing tissue putrescine with the aid of 5-fluoromethylornithine, a specific inhibitor of ornithine transaminase and hence the catabolism of L-ornithine. As a result of the treatment with the latter drug, the concentration of putrescine was further increased by a factor of 2-3 without any changes in the concentrations of spermidine and spermine. In the testis of transgenic mice treated with 5-fluoromethylornithine, the concentration of putrescine was nearly 60 times that in non-transgenic untreated animals, yet the concentration of spermidine was only 1.5-fold higher. A similar small increase in brain spermidine was accompanied by a 40-fold elevation in the concentration of putrescine. The apparent blockade between putrescine and spermidine was in all likelihood not attributable to an inhibition of S-adenosylmethionine decarboxylase, the rate-controlling enzyme in the biosynthesis of spermidine and spermine. Our results are more compatible with the view that in non-dividing adult tissues putrescine is sequestered through some unknown mechanisms in a way that makes it unavailable for the synthesis of the higher polyamines.

scholarly journals Transgenic mice expressing the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter

1996 ◽  
Vol 314 (2) ◽  
pp. 405-408 ◽  
Author(s):  
Leena ALHONEN ◽  
Sami HEIKKINEN ◽  
Riitta SINERVIRTA ◽  
Maria HALMEKYTÖ ◽  
Pekka ALAKUIJALA ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Ornithine Decarboxylase ◽  
Mammalian Cells ◽  
Fold Increase ◽  
Transgenic Animals ◽  
Decarboxylase Activity ◽  
Transgenic Mouse Line ◽  
Regulatory Machinery ◽  
Dividing Cells ◽  
Very High

We have generated a transgenic mouse line harbouring the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter. Even in the absence of an exposure to heavy metals, ornithine decarboxylase was over-expressed in heart, testis, brain, and especially in liver, of the transgenic animals. An exposure of the transgenic mice to zinc further enhanced the enzyme activity to a level which in liver represented up to 8000-fold increase in comparison with non-transgenic animals. The striking stimulation of liver ornithine decarboxylase activity upon treatment of the transgenic mice with zinc was accompanied by a nearly 150-fold increase in the hepatic putrescine content as compared with similarly treated non-transgenic animals. Even though the liver putrescine concentration reached that of spermidine and spermine in the transgenic animals, the contents of the higher polyamines only transiently increased upon zinc administration and then returned to the basal level. These findings once again indicate that mammalian cells possess extremely powerful regulatory machinery to prevent an over-accumulation of spermidine and spermine in non-dividing cells, and that very high tissue putrescine concentrations can be tolerated, at least for periods of a few days, with seemingly no phenotypic changes.

Hemostasis and coagulation at a hematocrit level of 0.85: functional consequences of erythrocytosis

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4416-4422 ◽  
Author(s):  
Junpei Shibata ◽  
Jo Hasegawa ◽  
Hans-Joachim Siemens ◽  
Eva Wolber ◽  
Leif Dibbelt ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Thrombus Formation ◽  
Transgenic Animals ◽  
Blood Analysis ◽  
Independent Manner ◽  
Coagulation Activity ◽  
Transgenic Mouse Line ◽  
Human Erythropoietin ◽  
Functional Consequences ◽  
Plasmatic Coagulation

Abstract We have generated a transgenic mouse line that reaches a hematocrit concentration of 0.85 due to constitutive overexpression of human erythropoietin in an oxygen-independent manner. Unexpectedly, this excessive erythrocytosis did not lead to thrombembolic complications in all investigated organs at any age. Thus, we investigated the mechanisms preventing thrombembolism in this mouse model. Blood analysis revealed an age-dependent elevation of reticulocyte numbers and a marked thrombocytopenia that matched the reduced megakaryocyte numbers in the bone marrow. However, platelet counts were not different from wild-type controls, when calculations were based on the distribution (eg, plasma) volume, thereby explaining why thrombopoietin levels did not increase in transgenic mice. Nevertheless, bleeding time was significantly increased in transgenic animals. A longitudinal investigation using computerized thromboelastography revealed that thrombus formation was reduced with increasing age from 1 to 8 months in transgenic animals. We observed that increasing erythrocyte concentrations inhibited profoundly and reversibly thrombus formation and prolonged the time of clot development, most likely due to mechanical interference of red blood cells with clot-forming platelets. Transgenic animals showed increased nitric oxide levels in the blood that could inhibit vasoconstriction and platelet activation. Finally, we observed that plasmatic coagulation activity in transgenic animals was significantly decreased. Taken together, our findings suggest that prevention of thrombembolic disease in these erythrocytotic transgenic mice was due to functional consequences inherent to increased erythrocyte concentrations and a reduction of plasmatic coagulation activity, the cause of which remains to be elucidated.

scholarly journals Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice.

1994 ◽  
Vol 180 (3) ◽  
pp. 861-872 ◽  
Author(s):  
R Cibotti ◽  
J P Cabaniols ◽  
C Pannetier ◽  
C Delarbre ◽  
I Vergnon ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Transgenic Mice ◽  
Transgenic Animals ◽  
Cell Receptor ◽  
T Cell Response ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Blood Levels ◽  
Transgenic Mouse Line ◽  
Hen Egg

We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.

Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression

2007 ◽  
Vol 97 (01) ◽  
pp. 99-108 ◽  
Author(s):  
Yuxi Feng ◽  
Franziska vom Hagen ◽  
Frederick Pfister ◽  
Snezana Djokic ◽  
Sigrid Hoffmann ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Mouse Retina ◽  
Vascular Density ◽  
Wild Type ◽  
Transgenic Mouse Line ◽  
Oxygen Induced Retinopathy ◽  
Genes Encoding ◽  
Angiopoietin 2 ◽  
The Impact

SummaryAngiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14 %; p<0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26 %; p<0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

The Hox a Locus Is Strongly Targeted by MMLV in an E2a-PBX1 Induced B-Cell Leukemia Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1529-1529
Author(s):  
Janet J. Bijl ◽  
Martin Sauvageau ◽  
Alex Thompson ◽  
Guy Sauvageau
Keyword(s):  
Transgenic Mice ◽  
B Cell ◽  
Gene Activation ◽  
Transgenic Animals ◽  
Inverse Pcr ◽  
Pcr Analysis ◽  
Cell Leukemia ◽  
Integration Sites ◽  
B Cell Leukemia ◽  
Control Tumor

Abstract We previously developed a B-ALL model from E2a-PBX1 transgenic mice (Bijl et al. Abstract 469 ASH 2003 and paper in preparation). We now exploit this model to identify oncogenic collaborators to E2a-PBX1. To achieve this, 18 newborn E2a-PBX1 transgenic mice and 23 control littermates were intraperitonally injected with MMLV. The occurrence of B-ALL in E2a-PBX1 transgenic animals was significantly accelerated when compared to control littermates (mean survival 162 ± 31 versus 191 ± 50 days, respectively, P=0.03), suggesting the presence of E2a-PBX1 specific collaborators. Inverse PCR was performed on genomic DNA isolated from seven E2a-PBX1 and six control tumors. Seventy-two different retroviral insertion sites were recovered from the tumors. Six common integration sites (CIS) for MMLV were identified; including two novel CIS, i.e. Pde4d, a phosphodiesterase, and a hypothetical phospholipase A930027K05Rik. Strikingly, the Hox a locus was targeted in six of seven E2a-PBX1 tumors, and only one of six control tumors. MMLV integrants in the Hox a locus were detected in a 19kb region located between Hoxa6/Hoxa7 and Hoxa10. Two tumors were targeted twice in this locus. Q-PCR analysis for expression of all Hox a cluster genes identified a significant increase for the Hoxa3 to Hoxa10 genes in the transgenic tumors when compared to the expression in a control MMLV induced B-cell tumor (see table, below). Hoxa7 and Hoxa6 were the most frequently and strongly activated (6/6 and 5/6 transgenic tumors, respectively), showing values of 5700 and 6600 fold over expression in one particular tumor. Interestingly, a Hox a locus insertion in a control tumor (table: last row) did not have any effect on gene activation. In this study we demonstrate a preference for MMLV to target the Hox a locus in an E2a-PBX1context. The resultant gene activation suggests that several members of the Hox a locus are functional collaborators to E2a-PBX1 in the induction of B-cell leukemia. Fold difference in expression for Hox a cluster genes in Hox a targeted tumors compared to a control tumor. Insertions a3 a4 a5 a6 a7 a9 a10 a9/a10 −1 1 4 3 138 2 4 a3 17 55 1067 5700 6608 171 62 a10 3 1 11 98 38 3 −4 a9/a10 1 −1 2 21 9 2 −2 a9/a10 2x 13 6 19 221 191 85 2 a7/a9 + a10 8 4 11 66 44 9 149 a10 −2 −6 2 2 −2 −8 −5

scholarly journals Transgenic Mice Overexpressing the Complement Inhibitor Crry as a Soluble Protein Are Protected from Antibody-induced Glomerular Injury

1998 ◽  
Vol 188 (7) ◽  
pp. 1321-1331 ◽  
Author(s):  
Richard J. Quigg ◽  
Chun He ◽  
Alice Lim ◽  
Dawn Berthiaume ◽  
Jessy J. Alexander ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Complex Disease ◽  
Alternative Pathway ◽  
Disease Model ◽  
Fractional Excretion ◽  
Transgenic Animals ◽  
Pcr Analysis ◽  
Glomerular Injury ◽  
Rt Pcr

Complement receptor 1–related gene/protein y (Crry) is a potent murine membrane complement regulator that inhibits classical and alternative pathway C3 convertases. In nephrotoxic serum (NTS) nephritis, injected antibodies (Abs) bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. To study the phenotypic effects of continuous complement pathway blockade, transgenic mice were created that express recombinant soluble (rs) Crry directed by the broadly active and heavy metal-inducible metallothionein-I promoter. One transgenic line expressing high levels of rsCrry was propagated. Serum rsCrry levels were 18.7 ± 2.7 μg/ml (n = 5) at basal level and increased to 118.1 ± 20.6 μg/ml 4 d after addition of zinc to the drinking water. By reverse transcription polymerase chain reaction (RT-PCR), transgene messenger (m)RNA was present in liver, kidney, brain, lung, and spleen, but not in heart. By in situ RT-PCR analysis of kidneys, transgene mRNA was widely expressed both in renal glomeruli and tubules. Urinary excretion of rsCrry was 113.4 ± 22.4 μg/ml with a fractional excretion relative to creatinine of 13.2 ± 2.7%, consistent with local renal production of rsCrry and secretion into urine. The founder and all transgene positive adult animals have remained healthy with no mortality or apparent phenotypic abnormalities, including infection or immune complex disease. To determine whether rsCrry blocked complement-mediated injury, NTS nephritis was induced by injection of NTS immunoglobulin (Ig)G, followed by an 18-h urine collection to quantitate the excretion of albumin as a measure of glomerular injury. In transgene-negative littermates (n = 15), transgene-positive animals (n = 10), and transgene-positive animals fed zinc (n = 10), albuminuria was 4,393 ± 948, 1,783 ± 454, and 1,057 ± 277 μg/mg creatinine, respectively (P &lt; 0.01 by ANOVA). Glomerular C3 was evident by immunofluorescence staining in 12/15 transgene-negative animals, but in none of the transgene-positive animals fed zinc. Thus, we have produced the first transgenic animals that overexpress a soluble C3 convertase inhibitor. rsCrry expression markedly ameliorates an Ab-induced disease model in vivo. These results support the hypothesis that continuous complement inhibition at the C3 convertase step is feasible and effective in complement-mediated injury states.

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

1991 ◽  
Vol 278 (3) ◽  
pp. 895-898 ◽  
Author(s):  
M Halmekytö ◽  
L Alhonen ◽  
J Wahlfors ◽  
R Sinervirta ◽  
T Eloranta ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Ornithine Decarboxylase ◽  
Transgenic Animals ◽  
Metabolizing Enzymes ◽  
Transgenic Mouse Line ◽  
Adenosylmethionine Decarboxylase ◽  
Transgenic Lines ◽  
Polymerase Chain

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.

scholarly journals Transgenic mice overexpressing ornithine and S-adenosylmethionine decarboxylases maintain a physiological polyamine homoeostasis in their tissues

1997 ◽  
Vol 323 (2) ◽  
pp. 457-462 ◽  
Author(s):  
Ritva HELJASVAARA ◽  
Ildiko VERESS ◽  
Maria HALMEKYTÖ ◽  
Leena ALHONEN ◽  
Juhani JÄNNE ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Ornithine Decarboxylase ◽  
Metabolic Flux ◽  
Pulse Labelling ◽  
Spermidine Synthase ◽  
Tissue Concentrations ◽  
Adenosylmethionine Decarboxylase ◽  
Normal Limits ◽  
Primary Fibroblasts ◽  
Hybrid Mice

Recent work has shown that transgenic mice overexpressing human ornithine decarboxylase display no marked changes in the tissue concentrations of spermidine or spermine in spite of a dramatic increase in putrescine levels. In the tissues of transgenic mice carrying the human spermidine synthase gene and in those of hybrid mice overexpressing both ornithine decarboxylase and spermidine synthase, spermidine and spermine levels remain within normal limits. To test whether the amount of the propylamine group donor, decarboxylated S-adenosylmethionine, limits the conversion of putrescine into the higher polyamines, we have produced transgenic mouse lines harbouring the rat S-adenosylmethionine decarboxylase gene in their genome. However, neither these mice nor the hybrid mice overexpressing both ornithine decarboxylase and S-adenosylmethionine decarboxylase displayed significant changes in their spermidine and spermine tissue levels. To study the mechanism by which cells maintain the constancy of the polyamine concentrations, we have determined the metabolic flux of polyamines in transgenic primary fibroblasts using pulse labelling. The results indicate that the polyamine flow is faster in transgenic primary fibroblasts than in non-transgenic fibroblasts and that the intracellular homoeostasis of higher polyamines is maintained at least partly by the acetylation of spermidine and spermine and their secretion into the medium.

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