New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

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 GFP-expression. 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.

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair.

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

[Vascular beta-adrenergic remodeling in rat transgenic model over-expressing endothelial beta3-adrenoceptors]. / Remodelage des récepteurs beta-adrénergiques vasculaires induit par la surexpression endothéliale du récepteur beta3-adrénergique dans un modèle de rat transgénique.

In rat thoracic aorta, the stimulation of endothelial beta3-adrenoceptors (beta-AR) produces a vasorelaxation through activation of a NO synthase pathway and an increase in cGMP levels. In hypertension, a global decrease of the beta-AR response has been described. In spontaneously hypertensive rats (SHR), we have shown that beta3-adrenoceptor-mediated relaxation was not modified in SHR aorta at the age of 12 weeks, in spite of an upregulation of beta3-adrenoceptors. In order to determine the consequences of an over-expression of the beta3-AR, we have developed a transgenic rat over-expressing specifically in endothelial cells the human beta3-AR (Tg beta3). By real-time quantitative PCR, we have determined the expression level of the different beta-AR subtypes. We confirmed an over-expression of the beta3-AR transcripts in Tg beta3 (ratio = 3.39 +/- 0.8; n=3 for Tg beta3 vs wild type [WT] animals). Surprisingly, we observed in Tg beta3 a decrease of beta1-AR transcripts (ratio = 0.76 +/- 0.03; n=3 for Tg beta3 vs WT animals) and no variation for beta2-AR transcripts (ratio = 1.95 +/- 0.60; n=3 for Tg beta3 vs WT animals). In aorta rings from WT and Tg beta3, the isoproterenol-induced relaxation was similar (WT: Emax = 82 +/- 6%, n=6; Tg beta3: Emax = 85 +/- 6, n=6). By contrast, in the presence of 10 microM nadolol, a beta1-, beta2-AR antagonist, the isoproterenol-induced response was significantly increased in Tg beta3 (WT: Emax = 68 +/- 6%, n=6: Tg beta3: Emax = 86 +/- 3; p < 0.01 vs WT). This effect was loss on denuded aortic rings. In conclusion, our study reported similar results to those obtained in hypertension in which a decrease of the beta-AR expression was associated to an elevation of the beta3-AR density. Moreover, this over-expression in our transgenic model is associated to a potential response induced by beta3-AR. Therefore, an activation of beta3-AR could supply the beta1- / beta2-AR decrease. Then, our transgenic model should be used to characterize the physiological consequences of this over-expression as well as to determine the putative involvement of this receptor in the pathogenesis of hypertension.

Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes.

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes. When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P<0.05) and neither was affected by CRISPR/Cas9 content in the injected medium. Embryo development to blastocyst was not affected by microinjection and was similar among the experimental groups. From 20 embryos analyzed by Sanger sequencing, ten were mutant (heterozygous or mosaic; 50% efficiency). To obtain live MSTN KO lambs, 53 blastocysts produced after zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and livestock.

Anti-adenovirus immune responses in rats are enhanced by interleukin 4 but not interleukin 10 produced by recombinant adenovirus.

Recombinant adenoviruses can be used for in vivo gene transfer with great efficiency. However, the duration of transgene expression and the possibility of readministering the virus are severely limited by the host anti-adenovirus immune response, which is controlled mainly by cytokine networks. Adenoviruses encoding IL-4 (AdIL-4) or IL-10 (AdIL-10) were administered to rats through the portal vein and the anti-adenovirus immune response was studied. As compared with administering adenoviruses without transgene (Addl324) or with the lacZ gene (AdlacZ), AdIL-4, but not AdIL-10, resulted in a significant increase in leukocytes in the liver, with a predominance of macrophages that peaked on days 7 and 14 after gene transfer and gradually returned to normal by day 28. AdIL-4 induced a significant increase in both neutralizing and ELISA-detected anti-adenovirus antibodies, whereas AdIL-10 caused an increase in ELISA-detected antibodies alone. Anti-adenovirus antibodies were predominantly of Th1-dependent immunoglobulin subclasses in rats receiving Addl324, AdlacZ, or AdIL-10, whereas animals receiving AdIL-4 showed a predominance of Th2-dependent immunoglobulin subclasses. Type 1 (IFN-gamma) and type 2 (IL-5) cytokines were increased only in livers from rats receiving AdIL-4. Rats receiving AdIL-4 showed increased anti-adenovirus cytotoxic T lymphocyte activity and CD8+ cell depletion prevented leukocyte infiltration in the liver. These results show that IL-4 increases local and systemic immune responses against recombinant adenoviruses.

Genetic engineering in allotransplantation of vascularized organs.

Transplantation offers a unique opportunity for gene transfer into allografts before grafting. After organ retrieval, the cold ischemic period renders organs available for manipulation and gene transfer. Local expression of protective or immunomodulatory molecules within the graft environment offers a better local bioavailability of bioreagents and potentially less systemic side effects. Protection against ischemia-reperfusion injury, acute and/or chronic rejection without significant side effects would be a major breakthrough in transplant research. However, protocols of transfection adapted to the transplant setting and control of gene expression must be clearly evaluated before going to clinical trials. The first part of this review deals with gene transfer techniques into the allograft, emphasizing particular transplant conditions that are encountered and that must be respected when designing protocols for gene transfer experiments. The second part deals with specific therapeutic strategies to protect and prolong allograft survival.

Protection of rat endothelial cells from primate complement-mediated lysis by expression of human CD59 and/or decay-accelerating factor.

The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.

Inhibition of graft arteriosclerosis development in rat aortas following heme oxygenase-1 gene transfer.

Heme oxygenase 1 (HO-1) is an enzyme which degrades heme into tree end products: biliverdin, free iron and carbon monoxide. This enzyme has recently been shown to have anti-inflammatory and tissue protective effects. HO-1 expression is involved in organ protection in pathological situations, and immunosuppressive treatments resulting in indefinite graft survival without chronic rejection have been associated with HO-1 expression by cells of the vessel wall. The aim of this study was to analyze the effect of specific HO-1 overexpression. We used a recombinant adenovirus coding for human HO-1 cDNA in a rat aorta chronic rejection model, 30 days after transplantation. Control groups included rats non treated or treated with a non-coding adenovirus Addl324. We first demonstrated that AdHO-1 was efficiently expressed in endothelial cells in vitro, and in rat aortas ex vivo after adenovirus gene transfer. We found that intimal thickening in AdHO-1 treated aortas (10.8 +/- 3.8%, n=5) was significantly decreased compared to untreated (21.2 +/- 5.6%, n = 5) or Addl324-treated (21.1 +/- 1.2%, n = 4) aortas. Immunohistology showed that treatment with AdHO-1 resulted in a significant reduction in leukocyte infiltration and a decreasing number of VSMC in the intima, compared to Addl324-treated aortas. However, this effect of HO-1 on chronic rejection did not imply modifications on numbers of apoptotic cells in the graft or of alloantibody levels. We have demonstrated, for the first time, that specific HO-1 overexpression following gene transfer of HO-1 inhibited chronic rejection by reducing leukocyte and VSMC infiltration of the aorta intima.

Generation of heme oxygenase-1-transgenic rats.

Heme oxygenase-1 (HO-1) expression protects cells from a variety of cellular insults and inhibits inflammation. However, its role in the regulation of immune responses has not yet been clearly established. We generated HO-1 transgenic rats to directly test the impact of HO-1 on the different immune mechanisms. To temporally control the expression of HO-1, we used a one-plasmid tetracycline (tet)-inducible system. This plasmid contains the H-2K(b) promoter, which transcribes the tet transactivator (tTA) and expression of a human HO-1 cDNA is obtained in the absence of tetracycline. The DNA construct was microinjected into one-cell rat embryos and mothers and pups were maintained with tetracycline. Eight transgenic founders were obtained. Analysis of transgene expression in the absence of tet showed that 2 lines (12.4 and 12.6) expressed HO-1 mRNA in several organs (as detected by reverse transcription polymerase chain reaction) and at the protein level only in the thymus. Expression levels of transgene-derived HO-1 increased after withdrawal of tet compared with transgenic rats maintained with tet, as detected by analysis of mRNA levels by quantitative real-time reverse transcription polymerase chain reaction. Gross examination and histopathological analysis of several organs in both lines showed no anomalies. Thymocytes and splenocytes of both lines showed normal cell subpopulations and allogeneic proliferation compared with controls. Systemic immune responses against cognate antigens were normal in both lines, as evaluated by the proliferation of lymph node cells and the production of antibodies against keyhole limpet hemocyanin after immunization. Animals from line 12.6 rejected transplanted allogeneic hearts with the same kinetics as controls. In conclusion, short-term induction of HO-1 overexpression did not modify immune responses compared to those of control non-transgenic animals.

Stilbene content of mature Vitis vinifera berries in response to UV-C elicitation.

A method using HPLC analysis has been used to compare the level of resveratrol and its derivatives, piceid, pterostilbene and epsilon-viniferin, in grapevine berries of three Vitis vinifera varieties. The concentration of these compounds has been evaluated in healthy and Botrytis cinerea infected grape clusters, both in natural vineyard conditions and in response to UV elicitation.

[Protective effect of an apoptosis inhibitor in a new model of hepatitis induced by interleukin-4 in the rat]. / Effet protecteur d'un peptide inhibiteur de l'apoptose dans un nouveau modèle d'hépatite induite par l'interleukine-4 chez le rat.

OBJECTIVES: Interleukin-4 is a cytokine with pleiotropic effects on many cells. The effects of its expression on the liver remain unclear. To obtain organ-localized cytokine expression and analyze its effect on the liver, recombinant adenovirus with coding sequences of interleukin-4 were transduced to rat livers. METHODS: Adenovirus with coding sequences of rat interleukin-4 were injected into the portal vein of Wistar rats. Microscopic examination of the liver was performed. The effects of interleukin-4 were confirmed in vitro on primary cultured rat hepatocytes. The same analysis was performed after intraperitoneal injection of l'YVADcmk, an inhibitor of the interleukin 1 converting enzyme. RESULTS: Interleukin-4 expression due to the recombinant adenovirus produced dose-related, potentially lethal, severe hepatitis. This hepatitis was characterized by a leucocyte infiltrate mainly composed of eosinophilic polymorphonuclear and mast cells with numerous apoptotic hepatocytes. Intraperitoneal injection of YVADcmk decreased hepatocyte apoptosis and biological hepatitis and prevented death. CONCLUSION: These results suggested that YVADcmk might be used in fulminant hepatitis in which apoptosis is predominant.

Usefulness of linkage disequilibrium of KM-19 and XV-2c DNA probes for genetic counselling in a high-risk CF family.

A French couple with an individual risk of carrying the cystic fibrosis (CF) mutation of 1/2 sought genetic counselling. From the DNA haplotypes generated by XV-2c and KM-19 RFLPs, it could be deduced that only one subject was a carrier, lowering the risk of having a CF baby from 1/16 to 1/200. The strong linkage disequilibrium between these RFLPs and the CF allele observed in France reduced the risk to 1/1600.

Analysis of human CD59 tissue expression directed by the CMV-IE-1 promoter in transgenic rats.

The investigation of human complement (C) inhibitors with a view to overcoming C-mediated tissue injury stands to benefit from the production of anatomically suitable transgenic animals. In this study, we used the CMV-IE1 enhancer/promoter to control the expression in vivo in transgenic rats of the human terminal C protein inhibitor CD59. Five transgenic rats were identified, of which four possessed at least one complete copy of the transgene. The presence of human CD59 transcripts and protein was demonstrated in two transgenic rat lines. A widespread tissue distribution of cells expressing human CD59, similar in the two lines, was observed-principally in pancreas, brain, heart, kidney, intestine and striated muscle. Whereas expression in pancreas and brain was uniform, mosaicism of CD59 expression was observed in some tissues such as heart and kidney, a proportion of cells within the tissue not expressing the transgene. Immunohistological analysis revealed surface expression of human CD59 in a variety of cells, including fibroblasts, epithelial cells and muscle cells, but not in endothelial cells. In conclusion, this paper analyses at the cellular level human CD59 expression directed by the CMV promoter in transgenic rats, amd discusses how they could be used to investigate in vivo the role of C in a variety of pathologies.

Frequency of the major CF mutation in French CF patients.

We have studied 124 patients of French origin, whose CF status had already been clearly established. These children belong to families previously tested with restriction fragment length polymorphism (RFLP) markers in our laboratory for genetic counselling. The most common mutation (delta F508) accounts for 67% in this population sample.