Expression of mitofusin 2(R94Q) in a transgenic mouse leads to Charcot-Marie-Tooth neuropathy type 2A.

Charcot-Marie-Tooth disease type 2A is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene. Mitofusin 2 encodes a mitochondrial outer membrane protein that participates in mitochondrial fusion in mammalian cells. How mutations in this protein lead to Charcot-Marie-Tooth disease type 2A pathophysiology remains unclear. We have generated a transgenic mouse expressing either a mutated (R94Q) or wild-type form of human mitofusin 2 in neurons to evaluate whether the R94Q mutation was sufficient for inducing a Charcot-Marie-Tooth disease type 2A phenotype. Only mice expressing mitofusin 2(R94Q) developed locomotor impairments and gait defects thus mimicking the Charcot-Marie-Tooth disease type 2A neuropathy. In these animals, the number of mitochondria per axon was significantly increased in the distal part of the sciatic nerve axons with a diameter smaller than 3.5 microm. Importantly, the analysis of R94Q transgenic animals also revealed an age-related shift in the size of myelinated axons leading to an over-representation of axons smaller than 3.5 microm. Together these data suggest a link between an increased number of mitochondria in axons and a shift in axonal size distribution in mitofusin 2(R94Q) transgenic animals that may contribute to their neurological phenotype.

A critical lineage-nonspecific role for pTalpha in mediating allelic and isotypic exclusion in TCRbeta-transgenic mice.

Although it is well established that early expression of TCRbeta transgenes in the thymus leads to efficient inhibition of both endogenous TCRbeta and TCRgamma rearrangement (also known as allelic and "isotypic" exclusion, respectively) the role of pTalpha in these processes remains controversial. Here, we have systematically re-evaluated this issue using three independent strains of TCRbeta-transgenic mice that differ widely in transgene expression levels, and a sensitive intracellular staining assay that detects endogenous TCRVbeta expression in individual immature thymocytes. In the absence of pTalpha, both allelic and isotypic exclusion were reversed in all three TCRbeta-transgenic strains, clearly demonstrating a general requirement for pre-TCR signaling in the inhibition of endogenous TCRbeta and TCRgamma rearrangement. Both allelic and isotypic exclusion were pTalpha dose dependent when transgenic TCRbeta levels were subphysiological. Moreover, pTalpha-dependent allelic and isotypic exclusion occurred in both alphabeta and gammadelta T cell lineages, indicating that pre-TCR signaling can potentially be functional in gammadelta precursors. Finally, levels of endogenous RAG1 and RAG2 were not down-regulated in TCRbeta-transgenic immature thymocytes undergoing allelic or isotypic exclusion. Collectively, our data reveal a critical but lineage-nonspecific role for pTalpha in mediating both allelic and isotypic exclusion in TCRbeta-transgenic mice.

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation.

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.

Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

Background: The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells.Results: The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages.Conclusion: Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells.

A comparative study of T-cell receptor V beta usage in non-obese diabetic (NOD) and I-E transgenic NOD mice.

The non-obese diabetic (NOD) mouse is a model for the study of insulin-dependent diabetes mellitus (IDDM). Recently transgenic NOD mice have been derived (NOD-E) that express the major histocompatibility complex (MHC) class II I-E molecule. NOD-E do not become diabetic and show negligible pancreatic insulitis. The possibility pertained that NOD-E mice are protected from disease by a process of T-cell deletion or anergy. This paper describes our attempts to discover whether this was so, by comparing NOD and NOD-E mouse T-cell receptor V beta usage. Splenocytes and lymph node cells were therefore tested for their ability to proliferate in response to monoclonal anti-V beta antibodies. We were unable to show any consistent differences between NOD and NOD-E responses to the panel of antibodies used. Previously proposed V beta were shown to be unlikely candidates for deletion or anergy. T cells present at low frequency (V beta 5+) in both NOD and NOD-E mice were shown to be as capable of expansion in response to antigenic stimulation as were more frequently expressed V beta. Our data therefore do not support deletion or anergy as mechanisms which could account for the observed disease protection in NOD-E mice.

Traducción comentada de “Genetically Modified Organisms (GMOs): Transgenic Crops and Recombinant DNA Technology”

The purpose of this paper is to provide a translation into Spanish of a review articleabout genetically modified organisms (GMOs) entitled “Genetically ModifiedOrganisms (GMOs): Transgenic Crops and Recombinant DNA Technology” publishedby the well-known scientific journal Nature. In a world where English has become thelingua franca when it comes to transferring scientific knowledge and information, itmust be taken into account that not everyone—from scientist to the general public—hasa good enough command of English so that they can feel comfortable enough reading inthis language. Translators are consequently needed resulting from a great demand oftranslation activity into, for example, Spanish. This is the reason why the proposedSpanish translation is followed by a detailed analysis emphasizing the difficulties andproblems that characterize scientific—and also general—translation (i.e. terminology,syntax, semantics, pragmatics, and ideology), for which different approaches as how tosolve them are provided. On the basis of the analysis, it can be concluded thatexperience will be of much help to scientific translators, given that specificterminological knowledge and style requirements must always be born in mind whentranslating in this field. Moreover, this paper is intended to serve as a guide forTranslation students specializing in the field of science and the expectation is to helpthem make the right decisions when it comes to translating. However, it is clear that itcan only be thought of as an introduction that should be completed with further researchand documentation tasks in order to offer a complete reference tool: the ultimatehandbook of scientific translation.

Transgenic plant factories for the production of biopolymers and plateform chemicals

In the mid-to long-term, resource constraints will force society to cover a significant share of the future demand for fuels, materials and chemicals by renewable resources. This trend is already visible in the increasing conversion of carbohydrates and plant oils to fuels, chemicals, and polymers. In this perspective, we discuss current efforts and ideas to produce platform chemicals and polymers directly in transgenic plants.

A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice.

The high Km glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic beta-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic beta-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.

Silencing mutant ataxin-3 rescues motor deficits and neuropathology in machado-joseph disease transgenic mice.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein - ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

Blood pressure and renin-angiotensin system resetting in transgenic rats with elevated plasma Val5-angiotensinogen.

OBJECTIVE: : Increases in plasma angiotensinogen (Ang-N) due to genetic polymorphisms or pharmacological stimuli like estrogen have been associated with a blood pressure (BP) rise, increased salt sensitivity and cardiovascular risk. The relationship between Ang-N, the resetting of the renin-angiotensin system, and BP still remains unclear. Angiotensin (Ang) II-induced genetic hypertension should respond to lisinopril treatment. METHODS: : A new transgenic rat line (TGR) with hepatic overexpression of native (rat) Ang-N was established to study high plasma Ang-N. The transgene contained a mutation producing Val-Ang-II, which was measured separately from nontransgenic Ile-Ang-II in plasma and renal tissue. RESULTS: : Male homozygous TGR had increased plasma Ang-N (∼20-fold), systolic BP (ΔBP + 26 mmHg), renin activity (∼2-fold), renin activity/concentration (∼5-fold), total Ang-II (∼2-fold, kidney 1.7-fold) but decreased plasma renin concentrations (-46%, kidney -85%) and Ile-Ang-I and II (-93%, -94%) vs. controls. Heterozygous TGR exhibited ∼10-fold higher plasma Ang-N and 17 mmHg ΔBP. Lisinopril decreased their SBP (-23 vs. -13 mmHg in controls), kidney Ang-II/I (∼3-fold vs. ∼2-fold) and Ile-Ang-II (-70 vs. -40%), and increased kidney renin and Ile-Ang-I (>2.5-fold vs. <2.5-fold). Kidney Ang-II remained higher and renin lower in TGR compared with controls. CONCLUSION: : High plasma Ang-N increases plasma and kidney Ang-II levels, and amplifies the plasma and renal Ang-II response to a given change in renal renin secretion. This enzyme-kinetic amplification dominates over the Ang-II mediated feedback reduction of renin secretion. High Ang-N levels thus facilitate hypertension via small increases of Ang II and may influence the effectiveness of renin-angiotensin system inhibitors.

The expansion of 300 CTG repeats in myotonic dystrophy transgenic mice does not induce sensory or motor neuropathy.

Although many studies have been carried out to verify the involvement of the peripheral nervous system (PNS) in dystrophia myotonica (DM1) patients, the results remain controversial. The generation of DM1 transgenic mice displaying the human DM1 phenotype provides a useful tool to investigate the type and incidence of structural abnormalities in the PNS. In the present study, the morphological and morphometric analysis of semi-thin sections of sciatic and sural nerves, lumbar dorsal root ganglia (DRG) and lumbar spinal cords revealed that in DM1 transgenic mice carrying 300 CTG repeats, there is no change in the number and diameter of myelinated axons compared to wild type. Only a non-significant reduction in the percentage of thin myelinated axons was detected in electron micrographs of ultra-thin sciatic nerve sections. Analysis of the number of neurons did not reveal a loss in number of either sensory neurons in the lumbar DRG or motor neurons in the lumbar spinal cord in these DM1 mice. Furthermore, in hind limb muscle sections, stained with a neurofilament antibody and alpha-bungarotoxin, the intramuscular axon arborization appeared normal in DM1 mice and undistinguishable from that in wild-type mice. Moreover, in DM1 mice, there was no irregularity in the structure or an increase in the endplate area. Also statistical analysis did not show an increase in endplate density or in the concentration of acetylcholine receptors. Altogether, these results suggest that 300 CTG repeats are not sufficient to induce axonopathy, demyelination or neuronopathies in this transgenic mouse model.

Analysis of photoreceptor abnormality in GUCY2D E837D/R838S transgenic pigs

Purpose: We generated genetically engineered pigs expressing the human dominant GUCY2DE837D/R838S allele to modelize cone dystrophy. After a functional follow-up showing reduced photopic ERG responses (ARVO 2011), we analyzed the eyes by immunohistochemistry and revealed retinal modifications in the transgenic group. Methods: Lentiviral vectors encoding the human double mutant GUCY2DE837D/R838S cDNA under the control of a portion of the pig arrestin-3 promoter (Arr3) were produced and used for lentiviral-mediated transgenesis in pigs. Animals were regularly submitted to behavioral and functional investigations and were sacrificed at 4, 7, 15 and 18 months of age for histological and RT-PCR analyses. Retinal markers were used to evaluate the retinal status of eleven transgenic pigs and 6 non-transgenic controls. The expression of the mutant cDNA was also assayed by RT-PCR. Results: A significant increase in the number of displaced nuclei in the outersegment layer is observed in transgenic animals compared to control animals independently of their age. Part of these nuclei originate from cones as demonstrated by colocalization with cone markers. No significant change in the ONL thickness (central and peripheral retina) was measured between 4 and 18 months of age, showing a slow progression of the disease in the transgenic pigs within this time-frame. Conclusions: Arr3-GUCY2DE837D/R838S pigs show signs of retinal abnormality with slow progression which parallels the loss of photopic function. Further characterization of this model should help to elucidate the molecular mechanisms underlying the disease evolution.

Transgenic mice and their impact on kidney research.

The kidney is a key organ in the maintenance of ion and fluid homeostasis and specific transport systems localized along the nephron guarantee this function. Due to its large functional heterogeneity, experiments on the whole organ level cannot be easily performed, and thus more refined tools are needed, like for example the development of specific recombination systems to gain knowledge on the physiological role of single proteins implicated in ion transport. This review introduces the transgenic technology developed over the past decades, and then focuses on recent strategies for generating kidney-specific gene targeting, over-expression, and gene ablation in mice, that will help to understand the physiological role of proteins implicated in salt and water balance in the kidney.