Séverine Remy - Academia.edu (original) (raw)

Papers by Séverine Remy

bioRxiv, 2021

Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal Na retention leadin... more Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal Na retention leading to oedema. This Na retention is usually attributed to epithelial sodium channel (ENaC) activation following plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped rat model of INS (CC-PAN), we showed that the observed electrogenic and amiloride-sensitive Na retention could not be attributed to ENaC. We, then, identified a truncated variant of acid sensing ion channel 2b (ASIC2b) that induced sustained acid-stimulated sodium currents when co-expressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that expression of the truncated ASIC2b in kidney was dependent on the presence of albumin in the tubule lumen and activation of ERK in renal cells. Finally, the presence of ASIC2 mRNA was also detected in kidney biopsies from patients with INS but in any of ...

In mammalian DNA cytosine methylation occurs specifically at CpG dinucleotide. Although the full ... more In mammalian DNA cytosine methylation occurs specifically at CpG dinucleotide. Although the full array of function of DNA methylation is yet to be elucidated, it is well established that DNA methylation is an important mechanism involved in gene expression, DNA replication and cancer. Rat glioma C6.9 cells undergo programmed cell death (PCD) after treatment with 1,25-dihydroxyvitamin D3 (1,25-D3). Hence, these cells were used to study whether DNA methylation was involved in the control of PCD. We found that 1,25-D3-mediated PCD of C6.9 cells was suppressed by exposure of the cells to the DNA demethylating agents 5-azacytidine (5-AzaC) and 5-aza-2%-deoxycytidine. This effect remains detectable several cell divisions following removal of 5-AzaC and, therefore, involves DNA methylation as an epigenetic regulatory mechanism of PCD. Accordingly, internucleosomal fragmentation, a feature of apoptosis that is detected in 1,25-D3-treated cells, is no longer observable after treatment of these cells with 5-AzaC. However, 5-AzaC does not totally suppress the responsiveness of C6.9 cells to 1,25-D3 since the induction of the c-myc gene remains unaffected. These results suggest that a change in DNA methylation pattern could suppress 1,25-D3-mediated PCD through the expression of previously hypermethylated genes such as proto-oncogenes with death-repressor activity, endogenous virus sequences or even genes inducing change in the differentiated state of these cells.

Journal of Leukocyte Biology

Frontiers in Genetics

The rat has been extensively used as a small animal model. Many genetically engineered rat models... more The rat has been extensively used as a small animal model. Many genetically engineered rat models have emerged in the last two decades, and the advent of gene-specific nucleases has accelerated their generation in recent years. This review covers the techniques and advances used to generate genetically engineered rat lines and their application to the development of rat models more broadly, such as conditional knockouts and reporter gene strains. In addition, genome-editing techniques that remain to be explored in the rat are discussed. The review also focuses more particularly on two areas in which extensive work has been done: human genetic diseases and immune system analysis. Models are thoroughly described in these two areas and highlight the competitive advantages of rat models over available corresponding mouse versions. The objective of this review is to provide a comprehensive description of the advantages and potential of rat models for addressing specific scientific questi...

American Journal of Respiratory Cell and Molecular Biology

Transplantation

In vivo analysis of human immune responses in immunodeficient rats.

Circulation Research

Rationale: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of fu... more Rationale: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K + channel, have been identified in pulmonary arterial hypertension patients. Objective: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. Methods and Results: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial ...

Duchenne muscular dystrophy (DMD) has as standard pharmacological therapy with corticoisteroids (... more Duchenne muscular dystrophy (DMD) has as standard pharmacological therapy with corticoisteroids (CS) that decrease inflammation and immune responses present in patients and animal models. CS have however limited efficacy and important and numerous side effects. Therefore, there is a need for new anti-inflammatory and pro-tolerogenic treatments that could replace or decrease doses of CS. We first assessed the status of immune system of dystrophin-deficient rats (Dmdmdx) that closely reproduce the phenotype of DMD patients. Dmdmdx rats showed increased leukocyte infiltration in skeletal and cardiac muscles, containing mostly macrophages but also T cells, and increased expression of several cytokines. Anti-CD45RC Monoclonal antibody (Mab) treatment induced immune tolerance in models of organ transplantation and GVHD (Graft Versus Host Disease). We observed that muscles and blood of DMD patients contained T CD4+ and CD8+ expressing high levels of CD45RChigh cells. Treatment of young Dmd...

Transplantation, Jan 24, 2018

Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunog... more Immunodeficient mice are invaluable tools to analyze the long-term effects of potentially immunogenic molecules in the absence of adaptive immune responses. Nevertheless, there are models and experimental situations that would beneficiate of larger immunodeficient recipients. Rats are ideally suited to perform experiments in which larger size is needed and are still a small animal model suitable for rodent facilities. Additionally, rats reproduce certain human diseases better than mice, such as ankylosing spondylitis and Duchenne disease and these disease models would greatly benefit of immunodeficient rats to test different immunogenic treatments. We describe the generation of Il2rg-deficient rats and their crossing with previously described Rag1-deficient rats to generate double-mutant RRG animals. As compared to Rag1-deficient rats, Il2rg-deficient rats were more immunodeficient since partially lacked not only T and B cells but also NK cells. RRG animals showed a more profound im...

Scientific reports, Jan 29, 2017

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection in... more The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.

Transgenic Research

On May 11th and 12th 2017 was held in Nantes, France, the international meeting &amp;amp;... more On May 11th and 12th 2017 was held in Nantes, France, the international meeting &amp;amp;amp;quot;Advances in transgenic animal models and techniques&amp;amp;amp;quot; ( http://www.trm.univ-nantes.fr/ ). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility ( http://www.tgr.nantes.inserm.fr/ ). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR François Bonamy, CNRS, Région Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting.

Journal of genetics and genomics = Yi chuan xue bao, May 4, 2016

The recent emergence and application of engineered endonucleases have led to the development of g... more The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the singl...

Scientific reports, Jan 7, 2015

The generation of genetically-modified organisms has been revolutionized by the development of ne... more The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRN...

Clinical Immunology, 2007

Methods in molecular biology (Clifton, N.J.), 2016

The rat is an important animal model to understand gene function and model human diseases. Since ... more The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

Scientific reports, Aug 17, 2016

BAC transgenic mammalian systems offer an important platform for recapitulating human gene expres... more BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC...

Transgenic Research, 2010

Adoptive cell transfer studies in regenerative research and identification of genetically modifie... more Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFPexpression. The ''high-expresser'' line (GFP high ) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP high -transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The ''low-expresser'' line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Circulation, 2015

P ulmonary arterial hypertension (PAH) is a rare disorder, with a prevalence of 15 to 50 patients... more P ulmonary arterial hypertension (PAH) is a rare disorder, with a prevalence of 15 to 50 patients per million in the population. It is characterized by remodeling of the precapillary pulmonary arteries, with endothelial cell dysfunction contributing to endothelial and pulmonary artery smooth muscle cell proliferation. This remodeling increases pulmonary vascular resistance and pulmonary arterial pressure (mean pulmonary arterial pressure ≥25 mm Hg and a pulmonary capillary wedge Background-The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. Methods and Results-In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2 Δ140Ex1/+ rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. Conclusions-EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

Journal of immunology (Baltimore, Md. : 1950), Jan 2, 2015

Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells c... more Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24(int)CD38(+)CD27(+)IgD(-)IgM(+/low) subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-β1-dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD(-)IgM(+/low) B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two...

Transgenic research, Jan 10, 2015