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

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.

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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

Biochemical Journal ◽

10.1042/bj3380311 ◽

1999 ◽

Vol 338 (2) ◽

pp. 311-316 ◽

Cited By ~ 17

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.

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

Biochemical Journal ◽

10.1042/bj2780895 ◽

1991 ◽

Vol 278 (3) ◽

pp. 895-898 ◽

Cited By ~ 24

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.

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Efficient Generation of Mice with Consistent Transgene Expression by FEEST

Scientific Reports ◽

10.1038/srep16284 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Lei Gao ◽

Yonghua Jiang ◽

Libing Mu ◽

Yanbin Liu ◽

Fengchao Wang ◽

...

Keyword(s):

Mouse Model ◽

Transgenic Mice ◽

Transgenic Mouse ◽

Mouse Models ◽

Transgene Expression ◽

Es Cells ◽

Mouse Line ◽

Transgenic Mouse Models ◽

Transgenic Mouse Line ◽

A Genome

Abstract Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale.

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Transgenic mice over-expressing the human spermidine synthase gene

Biochemical Journal ◽

10.1042/bj2930513 ◽

1993 ◽

Vol 293 (2) ◽

pp. 513-516 ◽

Cited By ~ 13

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.

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A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽

10.1093/function/zqab012 ◽

2021 ◽

Vol 2 (3) ◽

Cited By ~ 1

Author(s):

Nelly Redolfi ◽

Elisa Greotti ◽

Giulia Zanetti ◽

Tino Hochepied ◽

Cristina Fasolato ◽

...

Keyword(s):

Transgenic Mouse ◽

Fluorescent Protein ◽

Ex Vivo ◽

Brain Slices ◽

Resonance Energy ◽

Mouse Line ◽

Isolated Cells ◽

Genomic Locus ◽

Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

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