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Copy number variations (CNVs) of the human 16p11.2 locus are associated with several developmental/neurocognitive syndromes. Particularly, deletion and duplication of this genetic interval are found in patients with autism spectrum disorders, intellectual disability and other psychiatric traits. The high gene density associated with the region and the strong phenotypic variability of incomplete penetrance, make the study of the 16p11.2 syndromes extremely complex. To systematically study the effect of 16p11.2 CNVs and identify candidate genes and molecular mechanisms involved in the pathophysiology, mouse models were generated previously and showed learning and memory, and to some extent social deficits. To go further in understanding the social deficits caused by 16p11.2 syndromes, we engineered deletion and duplication of the homologous region to the human 16p11.2 genetic interval in two rat outbred strains, Sprague Dawley (SD) and Long Evans (LE). The 16p11.2 rat models displayed convergent defects in social behavior and in the novel object test in male carriers from both genetic backgrounds. Interestingly major pathways affecting MAPK1 and CUL3 were found altered in the rat 16p11.2 models with additional changes in males compared to females. Altogether, the consequences of the 16p11.2 genetic region dosage on social behavior are now found in three different species: humans, mice and rats. In addition, the rat models pointed to sexual dimorphism with lower severity of phenotypes in rat females compared to male mutants. This phenomenon is also observed in humans. We are convinced that the two rat models will be key to further investigating social behavior and understanding the brain mechanisms and specific brain regions that are key to controlling social behavior., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Martin Lorenzo, Muniz Moreno, Atas, Pellen, Nalesso, Raffelsberger, Prevost, Lindner, Birling, Menoret, Tesson, Negroni, Concordet, Anegon and Herault.)

Aims: Diastolic dysfunction is common in cardiovascular diseases, particularly in the case of heart failure with preserved ejection fraction. The challenge is to develop adequate animal models to envision human therapies in the future. It has been hypothesized that this diastolic dysfunction is linked to alterations in the nitric oxide ( • NO) pathway. To investigate this issue further, we investigated the cardiac functions of a transgenic rat model (Tgβ 3 ) that overexpresses the human β 3 -adrenoceptor (hβ 3 -AR) in the endothelium with the underlying rationale that the • NO pathway should be stimulated in the endothelium., Methods and Results: Transgenic rats (Tgβ 3 ) that express hβ 3 -AR under the control of intercellular adhesion molecule 2 promoter were developed for a specific expression in endothelial cells. Transcriptomic analyses were performed on left ventricular tissue from 45-week-old rats. Among all altered genes, we focus on • NO synthase expression and endothelial function with arterial reactivity and evaluation of • NO and O 2 •- production. Cardiac function was characterized by echocardiography, invasive haemodynamic studies, and working heart studies. Transcriptome analyses illustrate that several key genes are regulated by the hβ 3 -AR overexpression. Overexpression of hβ 3 -AR leads to a reduction of Nos3 mRNA expression (-72%; P < 0.05) associated with a decrease in protein expression (-19%; P < 0.05). Concentration-dependent vasodilation to isoproterenol was significantly reduced in Tgβ 3 aorta (-10%; P < 0.05), while • NO and O 2 •- production was increased. In the same time, Tgβ 3 rats display progressively increasing diastolic dysfunction with age, as shown by an increase in the E/A filing ratio [1.15 ± 0.01 (wild type, WT) vs. 1.33 ± 0.04 (Tgβ 3 ); P < 0.05] and in left ventricular end-diastolic pressure [5.57 ± 1.23 mmHg (WT) vs. 11.68 ± 1.11 mmHg (Tgβ 3 ); P < 0.05]. In isolated working hearts, diastolic stress using increasing preload levels led to a 20% decrease in aortic flow [55.4 ± 1.9 mL/min (WT) vs. 45.8 ± 2.5 mL/min (Tgβ 3 ); P < 0.05]., Conclusions: The Tgβ 3 rat model displays the expected increase in • NO production upon ageing and develops diastolic dysfunction. These findings provide a further link between endothelial and cardiac dysfunction. This rat model should be valuable for future preclinical evaluation of candidate drugs aimed at correcting diastolic dysfunction., (© 2020 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner., (© 2014 Remy et al.; Published by Cold Spring Harbor Laboratory Press.)

The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the alpha-tropomyosin pre-mRNA 3' end processing to the somite-specific pattern that results from the utilization of an upstream 3'-terminal exon designed exon 9A9'. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9' and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3' end processing of the alpha-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9', PTB regulates the cleavage/polyadenylation of this 3'-terminal exon.

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.

Currently, xenogeneic extracorporeal liver perfusion is used in the treatment of acute liver failure. In order to determine whether transgeneity for human regulatory proteins could improve the functional outcome of the ex-vivo liver in relation to the histopathological changes, we studied the effect of the humoral mechanism in xenogeneic isolated rat liver perfusion in normal and transgenic rat livers. Isolated rat liver perfusion was performed for 2 h in normal rat livers with Krebs Henseleit (KH) and human serum (HS), and in livers transgenic for human decay accelerating factor (hDAF; Tg HS). Function of the liver was established by measurement of liver enzymes and bile production, and clearance of bromosulphophthalein (BSP). Tissue specimens taken after perfusion were analysed by routine histology and immunofluorescence staining for C3c deposition. No change in release of liver enzymes could be established throughout the perfusion period. In the 2nd hour, a higher level of bile production was seen for the transgenic group than for the HS group. The transgenic rat livers outperformed the normal livers perfused with HS, when BSP concentration in the bile was measured; however, clearance of BSP from the perfusate was not significantly different. Haematoxylin-eosin (HE) staining of the liver tissue showed evidence of hyperacute rejection in the HS group. There was only mild tissue injury in the transgenic liver. High-intensity fluorescent staining for C3c deposition was seen in the normal livers perfused with HS, and significantly less in the transgenic livers. Although histologically less tissue damage and less C3c deposition was shown, no significantly improved function of the livers transgenic for hDAF was established. These results suggest that for short-term extracorporeal liver perfusion transgenesis offers no functional benefit.

Direct injection of naked DNA into skeletal or cardiac muscle induces detectable gene expression. Although this provides a practical system for transgene expression, the reported efficacy is too low to confer a therapeutic benefit. By following a rational strategy based on the supramolecular structures adopted by active complexes, we have discovered a novel nonionic amphiphile synthetic agent [poly(ethyleneoxide)(13)-poly(propyleneoxide)(30)-poly(ethyleneoxide)(13) block copolymer; PE6400] that enables gene expression in up to 35% of muscle fibers from mouse tibial cranial muscle. PE6400 abolishes the ceiling effect on transgene expression of increasing amounts of naked DNA and permits long-term expression of the beta-galactosidase reporter gene in immunologically tolerant transgenic rats. This improvement in gene expression over naked DNA was observed irrespective of the reporter gene, ranging from 0.7 to 3.4 kb, and of the animal model used. In skeletal muscle, the PE6400 formulation led to a level of transfection efficiency similar to that obtained by electrotransfer. PE6400 also promotes high transgene expression in cardiac muscle. In contrast, PE6400-DNA formulations were inefficient in vitro in established cell lines and in isolated cardiomyocytes. When microinjected into the cell cytoplasm, PE6400 promotes DNA trafficking into the nucleus and induces gene expression. PE6400 provides a simple gene delivery system for skeletal and myocardial gene transfer. We propose that the PE6400 formulation could serve for the treatment of diseases primarily affecting muscle or for the expression of therapeutic proteins for local or systemic benefit.

Background: Many patients with renal failure are condemned to long-term dialysis with little prospect of transplantation because they are highly sensitized with immunoglobulin G (IgG) directed against class I human leukocyte antigens (HLA) of virtually all donors. Xenotransplantation could represent an attractive solution providing their alloantibodies (alloAb) do not recognize porcine motifs. Hitherto there has been no in vivo demonstration of any cross-reactivity and the objective of this work was to investigate this problem using a technique of extracorporeal pig kidney perfusion as a model of clinical xenografting., Methods: Pig kidneys were perfused ex vivo with plasma from both a group of highly sensitized patients and healthy individuals. Sequential plasma samples were analyzed for the titer of anti-Galalpha1-3Gal antibody (Ab) (major natural xenoreactive Ab) by enzyme-linked immunosorbent assay and anti-HLA class I Ab against a cell panel. At the end of perfusion, kidneys were perfused with a citric acid buffer to elute bound Ab., Results: Galalpha1-3Gal Ab were shown to decrease rapidly in the plasma (in less than 10 min) and then reached a plateau. A fractional decrease in anti-HLA Ab was also found in some of the perfused plasma samples. Anti-Gal Ab were readily detected in all citric acid perfusates and anti-HLA Ab in 8 of 10. The HLA specificities of eluted Ab were mainly concordant with the originally designated specificities for each patient., Conclusion: Anti-HLA class I Ab presumably cross-react with pig class I homologues. However, some plasma samples did not cross-react, suggesting that negatively cross-matched pig kidneys could be identified in the pig population for xenotransplantation in these patients. Further studies are required to precisely describe these cross-reactivities and to understand their functional significance in xenotransplantation.

Specific tolerance to LEW.1W (RT1u) heart allografts can be induced in adult LEW.1A (RT1a) rats by donor-specific blood transfusion (DST). We have previously shown that both rejected and tolerated grafts are heavily infiltrated by T lymphocytes, and that in both cases these T cells are capable of developing similar cytotoxic responses against donor cells in vitro; tolerance is therefore not due to the deletion of alloreactive T cells. At the same time, we found that the accumulation of IL-2 and IFN-gamma mRNA was decreased in tolerated grafts compared with rejected grafts. These results suggested that the induction of allograft tolerance in DST-treated animals could be mediated by anergy or suppression of graft-infiltrating Th1 cells. Although Th1 and Th2 clones have not yet been characterized in the rat, peripheral CD4+ rat T cells can be divided into two populations, based on their expression of the isoform RC of the CD45 molecule. Upon activation, CD45RChigh CD4+ T cells produce IL-2 and IFN-gamma and responsible for the induction of the graft-versus-host reaction, whereas CD45RClow CD4+ T cells produce IL-4 in vitro and provide B cell help. In the present study, we show that heart allografts from both DST-treated and untreated rats were infiltrated by equivalent numbers of leukocytes, of which CD4+ T cells also made up similar percentages. Among these CD4+ T cells, we observed that in allografts from DST-treated recipients the CD45RChigh population on day 5 was very significantly smaller (P = 0.004) than in the untreated group, while CD45RClow populations remained comparable. Moreover, using a new quantitative RT-PCR method, we found a dramatic reduction in the accumulation of IL-2, IFN-gamma, IL-10, IL-4, and IL-13 mRNA in hearts from DST-treated recipients compared with those of untreated recipients during the week following transplantation. These results show that in heart allografts from DST-treated recipients, despite phenotypic changes suggesting Th1 inhibition by Th2 imbalance, T helper function was inhibited as a whole, and that in vivo the phenotype CD4+ CD45RClow does not always correlate with Th2-related cytokine-producing cells.

Permanent tolerance to allografts can be induced in adult rats by donor-specific transfusions (DST) prior to transplantation. We have previously reported, in a model of heart allograft, the presence of a heavy leukocyte infiltrate, in the allograft which displayed a strong allospecific cytotoxicity when tested in vitro against donor cells, and a strong accumulation of mRNA for granzyme A and perforin in vivo. In contrast, there was a major decrease in the accumulation of mRNA for interleukin-2 and interferon-gamma. These results suggested that the DST-induced tolerance was associated with a decrease in type-1 T helper (Th1) cell function. The major role of preformed antibodies in xeno and allorejection is clearly established. Nevertheless, the consequences of alloantibody production in acute rejection and tolerance induction remains to be elucidated. We here analyze the alloantibody response in rejecting and DST-treated recipients. We show that, after transplantation, tolerant recipients, in contrast to rejecting ones, mount a low IgM alloresponse that switches to low IgG production. Detailed analysis of IgG alloantibodies in DST-treated recipients revealed that their production decrease was not equally distributed. Whereas rejecting animals mounted a strong anti-class I and II IgG alloantibody response, DST-treated recipients produced anti-class II and low titers of anti-class I IgG alloantibodies. Furthermore, among IgG subclasses, tolerant recipients predominantly produced IgG2a, a profile which, in the rat, is compatible with a Th2-controlled response. Finally, the passive transfer of immune serum from rejecting animals to DST-treated recipients could abrogate the tolerance. We suggest that the absence of anti-class I alloantibodies combined with preserved and/or increased anti-class II production plays a major role in graft tolerance in this model. These results reinforced the role of alloantibodies in rejection and in induction of tolerance.