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During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 103 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.

Mammalian preimplantation development involves two lineage specifications: first, the CDX2-expressing trophectoderm (TE) and a pluripotent inner cell mass (ICM) are separated during blastocyst formation. Second, the pluripotent epiblast (EPI; expressing NANOG) and the differentiated primitive endoderm (PrE; expressing GATA6) diverge within the ICM. Studies in mice revealed that OCT4/POU5F1 is at the center of a pluripotency regulatory network. To study the role of OCT4 in bovine preimplantation development, we generated OCT4 knockout (KO) fibroblasts by CRISPR-Cas9 and produced embryos by somatic cell nuclear transfer (SCNT). SCNT embryos from nontransfected fibroblasts and embryos produced by in vitro fertilization served as controls. In OCT4 KO morulae (day 5), ∼70% of the nuclei were OCT4 positive, indicating that maternal OCT4 mRNA partially maintains OCT4 protein expression during early development. In contrast, OCT4 KO blastocysts (day 7) lacked OCT4 protein entirely. CDX2 was detected only in TE cells; OCT4 is thus not required to suppress CDX2 in the ICM. Control blastocysts showed a typical salt-and-pepper distribution of NANOG- and GATA6-positive cells in the ICM. In contrast, NANOG was absent or very faint in the ICM of OCT4 KO blastocysts, and no cells expressing exclusively NANOG were observed. This mimics findings in OCT4-deficient human blastocysts but is in sharp contrast to Oct4-null mouse blastocysts, where NANOG persists and PrE development fails. Our study supports bovine embryogenesis as a model for early human development and exemplifies a general strategy for studying the roles of specific genes in embryos of domestic species.

Arrhythmias are common and contribute substantially to cardiovascular morbidity and mortality. The underlying pathophysiology of arrhythmias is complex and remains incompletely understood, which explains why mostly only symptomatic therapy is available. The evaluation of the complex interplay between various cell types in the heart, including cardiomyocytes from the conduction system and the working myocardium, fibroblasts and cardiac immune cells, remains a major challenge in arrhythmia research because it can be investigated only in vivo. Various animal species have been used, and several disease models have been developed to study arrhythmias. Although every species is useful and might be ideal to study a specific hypothesis, we suggest a practical trio of animal models for future use: mice for genetic investigations, mechanistic evaluations or early studies to identify potential drug targets; rabbits for studies on ion channel function, repolarization or re-entrant arrhythmias; and pigs for preclinical translational studies to validate previous findings. In this Review, we provide a comprehensive overview of different models and currently used species for arrhythmia research, discuss their advantages and disadvantages and provide guidance for researchers who are considering performing in vivo studies.Various models of cardiac arrhythmia have been developed in several different animal species to study the mechanisms of disease. In this Review, Clauss and colleagues summarize the advantages and disadvantages of the models and species used in arrhythmia research and provide guidance to investigators planning experiments in this field.

Xenotransplantation of porcine organs has made remarkable progress towards clinical application. A key factor has been the generation of genetically multi-modified source pigs for xenotransplants, protected against immune rejection and coagulation dysregulation. While efficient gene editing tools and multi-cistronic expression cassettes facilitate sophisticated and complex genetic modifications with multiple gene knockouts and protective transgenes, an increasing number of independently segregating genetic units complicates the breeding of the source pigs. Therefore, an optimal combination of essential genetic modifications may be preferable to extensive editing of the source pigs. Here, we discuss the prioritization of genetic modifications to achieve long-term survival and function of xenotransplants and summarise the genotypes that have been most successful for xenogeneic heart, kidney, and islet transplantation. Specific emphasis is given to the choice of the breed/genetic background of the source pigs. Moreover, multimodal deep phenotyping of porcine organs after xenotransplantation into human decedents will be discussed as a strategy for selecting essential genetic modifications of the source pigs. In addition to germ-line gene editing, some of these modifications may also be induced during organ preservation/perfusion, as demonstrated recently by the successful knockdown of swine leukocyte antigens in porcine lungs during ex vivo perfusion.

The nucleus of a differentiated cell can be reprogrammed to a totipotent state by exposure to the cytoplasm of an enucleated oocyte, and the reconstructed nuclear transfer embryo can give rise to an entire organism. Somatic cell nuclear transfer (SCNT) has important implications in animal biotechnology and provides a unique model for studying epigenetic barriers to successful nuclear reprogramming and for testing novel concepts to overcome them. While initial strategies aimed at modulating the global DNA methylation level and states of various histone protein modifications, recent studies use evidence-based approaches to influence specific epigenetic mechanisms in a targeted manner. In this review, we describe—based on the growing number of reports published during recent decades—in detail where, when, and how manipulations of the epigenome of donor cells and reconstructed SCNT embryos can be performed to optimize the process of molecular reprogramming and the outcome of nuclear transfer cloning.

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin ( GTKO.hCD46.hTBM ), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications ( GTKO.hCD46.hTBM ) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days. Tweaking immune characteristics of donors and recipients could allow for successful cross-species organ transplantation. Here, the authors show that an anti-CD40 antibody therapy of baboons that received heart transplants from genetically modified pigs is key to their long-term survival.

Purpose of ReviewPorcine islets represent a potentially attractive beta-cell source for xenotransplantation into patients with type 1 diabetes, who are not eligible to islet allo-transplantation due to a lack of suitable human donor organs. Recent progress in genetic engineering/gene editing of donor pigs provides new opportunities to overcome rejection of xeno-islets, to improve their engraftment and insulin secretion capacity, and to reduce the risk for transmission of porcine endogenous retroviruses. This review summarizes the current issues and progress in islet xenotransplantation with special emphasis on genetically modified/gene edited donor pigs.Recent FindingsAttempts to overcome acute rejection of xeno-islets, especially after intraportal transplantation into the liver, include the genetic elimination of specific carbohydrate antigens such as αGal, Neu5Gc, and Sd(a) for which humans and—in part—non-human primates have natural antibodies that bind to these targets leading to activation of complement and coagulation. A complementary approach is the expression of one or more human complement regulatory proteins (hCD46, hCD55, hCD59). Transgenic attempts to overcome cellular rejection of islet xenotransplants include the expression of proteins that inhibit co-stimulation of T cells. Expression of glucagon-like peptide-1 and M3 muscarinic receptors has been shown to increase the insulin secretion of virally transduced porcine islets in vitro and it will be interesting to see the effects of these modifications in transgenic pigs and islet products derived from them. Genome-wide inactivation of porcine endogenous retrovirus (PERV) integrants by mutating their pol genes using CRISPR/Cas9 is a recent approach to reduce the risk for PERV transmission by xeno-islets.SummaryGenetic engineering/gene editing of xeno-islet donor pigs facilitated major progress towards clinical islet xenotransplantation. The required set of genetic modifications will depend on the source of islets (fetal/neonatal vs. adult), the mode of delivery (encapsulated vs. free), and the transplantation site.

‘Autosomal dominant tubulointerstitial kidney disease – UMOD ’ (ADTKD- UMOD ) is caused by impaired maturation and secretion of mutant uromodulin (UMOD) in thick ascending limb of Henle loop (TAL) cells, resulting in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). To gain insight into pathophysiology, we analysed proteome profiles of TAL-enriched outer renal medulla samples from ADTKD- UMOD and control mice by quantitative LC-MS/MS. In total, 212 differentially abundant proteins were identified. Numerous ER proteins, including BiP (HSPA5), phosphorylated eIF2α (EIF2S1), ATF4, ATF6 and CHOP (DDIT3), were increased abundant, consistent with UPR. The abundance of hypoxia-inducible proteins with stress survival functions, i.e. HYOU1, TXNDC5 and ERO1L, was also increased. TAL cells in ADTKD- UMOD showed a decreased proportion of mitochondria and reduced abundance of multiple mitochondrial proteins, associated with disturbed post-translational processing and activation of the mitochondrial transcription factor NRF1. Impaired fission of organelles, as suggested by reduced abundance of FIS1, may be another reason for disturbed biogenesis of mitochondria and peroxisomes. Reduced amounts of numerous proteins of the OXPHOS and citrate cycle pathways, and activation of the LKB1-AMPK-pathway, a sensor pathway of cellular energy deficits, suggest impaired energy homeostasis. In conclusion, our study revealed secondary mitochondrial dysfunction in ADTKD- UMOD.

The worldwide prevalence of diabetes mellitus and obesity is rapidly increasing not only in adults but also in children and adolescents. Diabetes is associated with macrovascular complications increasing the risk for cardiovascular disease and stroke, as well as microvascular complications leading to diabetic nephropathy, retinopathy and neuropathy. Animal models are essential for studying disease mechanisms and for developing and testing diagnostic procedures and therapeutic strategies. Rodent models are most widely used but have limitations in translational research. Porcine models have the potential to bridge the gap between basic studies and clinical trials in human patients. This article provides an overview of concepts for the development of porcine models for diabetes and obesity research, with a focus on genetically engineered models. Diabetes-associated ocular, cardiovascular and renal alterations observed in diabetic pig models are summarized and their similarities with complications in diabetic patients are discussed. Systematic multi-organ biobanking of porcine models of diabetes and obesity and molecular profiling of representative tissue samples on different levels, e.g., on the transcriptome, proteome, or metabolome level, is proposed as a strategy for discovering tissue-specific pathomechanisms and their molecular key drivers using systems biology tools. This is exemplified by a recent study providing multi-omics insights into functional changes of the liver in a transgenic pig model for insulin-deficient diabetes mellitus. Collectively, these approaches will provide a better understanding of organ crosstalk in diabetes mellitus and eventually reveal new molecular targets for the prevention, early diagnosis and treatment of diabetes mellitus and its associated complications.

Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45–52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro- and minidystrophins as well as utrophin, a protein with partially overlapping functions. With the advent of the CRISPR-Cas9-nuclease, genome editing offers a novel option of correction of the disease-cuasing mutations. Full restoration of the healthy gene by homology directed repair is a rare event. However, non-homologous end-joining (NHEJ) may restore the reading frame by causing exon excision. This approach has first been demonstrated in mice and then translated to large animals (dogs, pigs). This review discusses the potential opportunities and limitations of genome editing in DMD, including the generation of appropriate animal models as well as new developments in genome editing tools.