Xenotransplantation of porcine islet cells as a potential option for the treatment of type 1 diabetes in the future.

Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction [immediate blood-mediated inflammatory reaction (IBMIR)], we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.

Oncogenic KRAS signalling in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is almost universally fatal. The annual number of deaths equals the number of newly diagnosed cases, despite maximal treatment. The overall 5-year survival rate of <5% has remained stubbornly unchanged over the last 30 years, despite tremendous efforts in preclinical and clinical science. There is unquestionably an urgent need to further improve our understanding of pancreatic cancer biology, treatment response and relapse, and to identify novel therapeutic targets. Rigorous research in the field has uncovered genetic aberrations that occur during PDAC development and progression. In most cases, PDAC is initiated by oncogenic mutant KRAS, which has been shown to drive pancreatic neoplasia. However, all attempts to target KRAS directly have failed in the clinic and KRAS is widely assumed to be undruggable. This has led to intense efforts to identify druggable critical downstream targets and nodes orchestrated by mutationally activated KRAS. This includes context-specific KRAS effector pathways, synthetic lethal interaction partners and KRAS-driven metabolic changes. Here, we review recent advances in oncogenic KRAS signalling and discuss how these might benefit PDAC treatment in the future.

Polymorphisms in the promoter region of the adiponectin (ADIPOQ) gene are presumably associated with transcription level and carcass traits in pigs.

The main goal of this study was to screen for polymorphisms in the porcine adiponectin (ADIPOQ) gene promoter, analyse their influence on transcription and identify any association with production traits in pigs. A 1018-bp region of the ADIPOQ gene promoter was analysed in 113 pigs, and seven novel polymorphisms found. Luciferase assays were performed in HEK293 (human embryonic kidney) cells and primary porcine adipose mesenchymal stem cells (pADMSCs) to investigate their affect on promoter activity. A 16-bp indel (c.-106_-91delGCCAGGGGTGTGAGCC) was found to influence promoter strength in vitro. In the HEK293 cell line, the Del/Del genotype showed greater luciferase activity than did the Ins/Ins genotype (P < 0.01). In pADMSCs, the insertion genotype of the ADIPOQ promoter showed greater luciferase activity than did the deletion genotype (P < 0.01). An association study performed for two novel polymorphisms, c.-67G>A and the 16-bp indel, showed significant correlation with loin measurements in Polish Landrace (P < 0.05) and synthetic line 990 (P < 0.01) pigs.

Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep.

Eukaryotic cells contain two distinct genomes. One is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance. Cloning of mammals typically has been achieved via nuclear transfer, in which a donor somatic cell is fused by electoporation with a recipient enucleated oocyte. During this whole-cell electrofusion, nDNA as well as mtDNA ought to be transferred to the oocyte. Thus, the cloned progeny should harbour mtDNAs from both the donor and recipient cytoplasms, resulting in heteroplasmy. Although the confirmation of nuclear transfer has been established using somatic cell-specific nDNA markers, no similar analysis of the mtDNA genotype has been reported. We report here the origin of the mtDNA in Dolly, the first animal cloned from an established adult somatic cell line, and in nine other nuclear transfer-derived sheep generated from fetal cells. The mtDNA of each of the ten nuclear-transfer sheep was derived exclusively from recipient enucleated oocytes, with no detectable contribution from the respective somatic donor cells. Thus, although these ten sheep are authentic nuclear clones, they are in fact genetic chimaeras, containing somatic cell-derived nuclear DNA but oocyte-derived mtDNA.

Selective ablation of differentiated cells permits isolation of embryonic stem cell lines from murine embryos with a non-permissive genetic background.

Embryonic stem (ES) cells enable the engineering of precise modifications to the mouse genome by gene targeting. Although there are reports of cultured cell contributions to chimaeras in golden hamster, rat and pig, definitive ES cell lines which contribute to the germline have not been demonstrated in any species but mouse. Among mouse strains, genetic background strongly affects the efficiency of ES isolation, and almost all ES lines in use are derived from strain 129 (refs 1,4,5) or, less commonly, C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of CBA-derived ES lines. Hence, CBA mice may provide a convenient model of ES isolation in other species. In ES derivation it is critical that the primary explant be cultured for a sufficient time to allow multiplication of ES cell progenitors, yet without allowing extensive differentiation. Thus, differences in ES derivation between mouse strains may reflect differences in the control of ES progenitor cells by other lineages within the embryo. Here we describe a strategy to continuously remove differentiated cells by drug selection, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.

Cell-mediated transgenesis in rabbits: chimeric and nuclear transfer animals.

The ability to perform precise genetic engineering such as gene targeting in rabbits would benefit biomedical research by enabling, for example, the generation of genetically defined rabbit models of human diseases. This has so far not been possible because of the lack of functional rabbit embryonic stem cells and the high fetal and perinatal mortality associated with rabbit somatic cell nuclear transfer. We examined cultured pluripotent and multipotent cells for their ability to support the production of viable animals. Rabbit putative embryonic stem (ES) cells were derived and shown capable of in vitro and in vivo pluripotent differentiation. We report the first live born ES-derived rabbit chimera. Rabbit mesenchymal stem cells (MSCs) were derived from bone marrow, and multipotent differentiation was demonstrated in vitro. Nuclear transfer was carried out with both cell types, and embryo development was assessed in vitro and in vivo. Rabbit MSCs were markedly more successful than ES cells as nuclear donors. MSCs were transfected with fluorescent reporter gene constructs and assessed for nuclear transfer competence. Transfected MSCs supported development with similar efficiency as normal MSCs and resulted in the first live cloned rabbits from genetically manipulated MSCs. Reactivation of fluorescence reporter gene expression in reconstructed embryos was investigated as a means of identifying viable embryos in vitro but was not a reliable predictor. We also examined serial nuclear transfer as a means of rescuing dead animals.

HDAC2 mediates therapeutic resistance of pancreatic cancer cells via the BH3-only protein NOXA.

BACKGROUND: Although histone deacetylase inhibitors (HDACi) are promising cancer therapeutics regulating proliferation, differentiation and apoptosis, molecular pathways engaged by specific HDAC isoenzymes in cancer are ill defined. RESULTS: In this study we demonstrate that HDAC2 is highly expressed in pancreatic ductal adenocarcinoma (PDAC), especially in undifferentiated tumours. We show that HDAC2, but not HDAC1, confers resistance towards the topoisomerase II inhibitor etoposide in PDAC cells. Correspondingly, the class I selective HDACi valproic acid (VPA) synergises with etoposide to induce apoptosis of PDAC cells. Transcriptome profiling of HDAC2-depleted PDAC cells revealed upregulation of the BH3-only protein NOXA. We show that the epigenetically silenced NOXA gene locus is opened after HDAC2 depletion and that NOXA upregulation is sufficient to sensitise PDAC cells towards etoposide-induced apoptosis. CONCLUSIONS: In summary, our data characterise a novel molecular mechanism that links the epigenetic regulator HDAC2 to the regulation of the pro-apoptotic BH3-only protein NOXA in PDAC. Targeting HDAC2 will therefore be a promising strategy to overcome therapeutic resistance of PDAC against chemotherapeutics that induce DNA damage.

Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy.

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMDΔ52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMDΔ52 pigs1, intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. 2) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMDΔ51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMDΔ52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease.

Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts.

Ovine primary fetal fibroblasts were cotransfected with a neomycin resistance marker gene (neo) and a human coagulation factor IX genomic construct designed for expression of the encoded protein in sheep milk. Two cloned transfectants and a population of neomycin (G418)-resistant cells were used as donors for nuclear transfer to enucleated oocytes. Six transgenic lambs were liveborn: Three produced from cloned cells contained factor IX and neo transgenes, whereas three produced from the uncloned population contained the marker gene only. Somatic cells can therefore be subjected to genetic manipulation in vitro and produce viable animals by nuclear transfer. Production of transgenic sheep by nuclear transfer requires fewer than half the animals needed for pronuclear microinjection.

Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice.

The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene targeting in hypoxanthine phosphoribosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replaced by an HPRT minigene which was in turn replaced by human alpha-lactalbumin. The end result is a clean exchange of defined DNA fragments with no other DNA remaining at the target locus. Targeted ES cells at each stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible method of targeting specific alterations to animal genes. A typical strategy for future use would be to generate a null mutation which could then be used to produce multiple second-step alterations at the same locus.

Human-mouse interspecies collagen I heterotrimer is functional during embryonic development of Mov13 mutant mouse embryos.

To investigate whether the human pro alpha 1(I) collagen chain could form an in vivo functional interspecies heterotrimer with the mouse pro alpha 2(I) collagen chain, we introduced the human COL1A1 gene into Mov13 mice which have a functional deletion of the endogenous COL1A1 gene. Transgenic mouse strains (HucI and HucII) carrying the human COL1A1 gene were first generated by microinjecting the COL1A1 gene into wild-type mouse embryos. Genetic evidence indicated that the transgene in the HucI strain was closely linked to the endogenous mouse COL1A1 gene and was X linked in the HucII transgenic strain. Northern (RNA) blot and S1 protection analyses showed that the transgene was expressed in the appropriate tissue-specific manner and as efficiently as the endogenous COL1A1 gene. HucII mice were crossed with Mov13 mice to transfer the human transgene into the mutant strain. Whereas homozygous Mov13 embryos die between days 13 and 14 of gestation, the presence of the transgene permitted apparently normal development of the mutant embryos to birth. This indicated that the mouse-human interspecies collagen I heterotrimer was functional in the animal. The rescue was, however, only partial, as all homozygotes died within 36 h after delivery, with signs of internal bleeding. This could have been due to a functional defect in the interspecies hybrid collagen. Extensive analysis failed to reveal any biochemical or morphological abnormalities of the collagen I molecules in Mov13-HucII embryos. This may indicate that there was a subtle functional defect of the interspecies hybrid protein which was not revealed by our analysis or that another gene has been mutated by the retroviral insertion in the Mov13 mutant strain.

A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli.

The increasing use of peptides as pharmaceutical agents, especially in the antiviral and anti-infective therapeutic areas, requires cost-effective production on a large scale. Many peptides need carboxy amidation for full activity or prolonged bioavailability. However, this modification is not possible in prokaryotes and must be done using recombinant enzymes or by expression in transgenic milk. Methods employing recombinant enzymes are appropriate for small-scale production, whereas transgenic milk expression is suitable for making complex disulfide-containing peptides required in large quantity. Here we describe a method for making amidated peptides using a modified self-cleaving vacuolar membrane ATPase (VMA) intein expression system. This system is suitable for making amidated peptides at a laboratory scale using readily available constructs and reagents. Further improvements are possible, such as reducing the size of the intein to improve the peptide yields (the VMA intein comprises 454 amino acids) and, if necessary, secreting the fusion protein to ensure correct N-terminal processing to the peptide. With such developments, this method could form the basis of a large-scale cost-effective system for the bulk production of amidated peptides without the use of recombinant enzymes or the need to cleave fusion proteins.

Germline gene polymorphisms predisposing domestic mammals to carcinogenesis.

Cancer is a complex disease caused in part by predisposing germline gene polymorphisms. Knowledge of carcinogenesis in companion mammals (dog and cat) and some livestock species (pig and horse) is quite advanced. The prevalence of certain cancers varies by breed in these species, suggesting the presence of predisposing genetic variants in susceptible breeds. This review summarizes the present understanding of germline gene polymorphisms, including BRCA1, BRCA2, MC1R, KIT, NRAS and RAD51, associated with predisposition to melanoma, mammary cancer, osteosarcoma and histiocytic sarcoma in dogs, cats, pigs and horses. The predisposing variants in these species are discussed in the context of human germline gene polymorphisms associated with the same types of cancer.

A porcine model of osteosarcoma.

We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53(R167H) and KRAS(G12D), and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7-8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease.

Method for producing transgenic bovine embryos from in vitro matured and fertilized oocytes.

Microinjection and in vitro culture procedures were developed to produce transgenic bovine embryos after in vitro fertilization of in vitro matured oocytes. In Experiment I, zygotes were subjected to pronuclear microinjection of DNA 18 or 24 h following addition of spermatozoa to oocytes. Microinjections were performed in either Hepes-buffered TCM-199 or modified Dulbecco's phosphate-buffered saline without glucose. Viability of embryos was similar at both injection times and for both media, as determined by morphological evaluation after culturing embryos in vitro for 10 d. In Experiment II, microinjected embryos were cultured 1) in rabbit oviducts, 2) in vitro in a 5% CO(2) in air, or 3) in a 5% CO(2) / 5% O(2) / 90% N(2) incubator. There were no significant differences between the 2 in vitro culture environments. The in vitro culture systems supported development of embryos significantly better than the rabbit oviducts; 33% of cleaved ova developed to blastocysts in vitro vs 10% in vivo; 98% of transferred ova were recovered from the rabbit oviducts. From both experiments, 6 of 92 blastocysts were positive for the microinjected DNA as determined by a polymerase chain reaction followed by gel electrophoresis.

Hybrid voltage sensor imaging of eGFP-F expressing neurons in chicken midbrain slices.

BACKGROUND: Dendritic computation is essential for understanding information processing in single neurons and brain circuits. Optical methods are suited best to investigate function and biophysical properties of cellular compartments at high spatial and temporal resolution. Promising approaches include the use of voltage sensitive dyes, genetically encoded voltage sensors, or hybrid voltage sensors (hVoS) consisting of fluorescent proteins and voltage-dependent quenchers that, so far, are not available in avian neuroscience. NEW METHOD: We have adapted a hVoS system for a chicken midbrain slice preparation by combining genetically expressed farnesylated eGFP with dipicrylamine (DPA). Depending on the cellular potential, DPA is shifted in the membrane, resulting in quenching of eGFP fluorescence linearly to the membrane potential by Förster resonance electron transfer. RESULTS: In ovo electroporation resulted in labelled neurons throughout the midbrain with a high level of fine structural detail. After application of DPA, we were able to optically record electrically evoked action potentials with high signal-to-noise ratio and high spatio-temporal resolution. COMPARISON WITH EXISTING METHODS: Standard methods available for avian neuroscience such as whole-cell patch clamp yield insufficient data for the analysis of dendritic computation in single neurons. The high spatial and temporal resolution of hVoS data overcomes this limitation. The results obtained by our method are comparable to hVoS data published for mammals. CONCLUSIONS: With the protocol presented here, it is possible to optically record information processing in single avian neurons at such high spatial and temporal resolution, that cellular and subcellular events can be analysed.

Polymorphism in 3' untranslated region of the pig PPARA gene influences its transcript level and is associated with adipose tissue accumulation.

The PPARA (peroxisome proliferator-activated receptor-α) gene encodes a nuclear receptor that plays an important role in fatty acid catabolism by transcriptional regulation of genes involved in fatty acid oxidation and can be considered as a candidate gene for fatness traits in the pig. The aim of the study was to search for a functional polymorphism in 3' untranslated region (UTR), their association with production traits, and postnatal PPARA transcript level in 2 skeletal muscles (longissimus and semimembranosus) of 5 commercial pig breeds (Polish Landrace [PL], Polish Large White [PLW], Duroc, Pietrain, and Pulawska). Altogether, 9 novel polymorphisms (8 SNP and 1 indel) were found in the 3' UTR. The in silico analysis revealed 6 putative microRNA target sequences in the analyzed region. The c.*636A>G substitution was widely distributed across breeds and located near the putative target sequence for miR-224. The relative PPARA transcript level was higher (P < 0.05) in LM of AA than in those of GG homozygous animals for SNP c.*636A>G. The luciferase assay revealed that miR-224 probably acts as a negative regulator of the PPARA expression in pig adipocytes (P = 2.9 × 10(-7)), but we did not observe the effect of the A or G alleles on the interaction between miR-224 and its putative target sequence. We hypothesize that the 2 predominant haplotypes, differing at 4 sites (including c.*636A>G), present different architecture of its 3' UTR and it could affect the level of the transcript. The c.*636A>G SNP, analyzed in PL and PLW, was significantly associated with backfat thickness at 3 points (P < 0.05) and intramuscular fat content (P < 0.01) in PL. Suggestive associations were found between 4 SNP (c.*321A>C, c.*324G>C, c.*626T>C, and c.*636A>G) and fatty acid contents in LM and subcutaneous and visceral fat tissue of PL, PLW, Duroc and Pietrain pigs. The PPARA mRNA level was higher in semimembranosus muscle than in LM (P = 8.38 × 10(-12)) in a general comparison and the same trend was found in most breeds (except for PL) and at all tested days of age (60, 90, 120, 150, 180, and 210 d). The effect of breed was highly significant in a general comparison (P = 0.48 × 10(-8)), but there was no common expression pattern in both muscles among different age groups. We conclude that the c.*636A>G SNP in the PPARA gene can be considered in PL breed as a useful genetic marker for adipose tissue accumulation.

Purification and Characterization of a Population of EGFP-Expressing Cells from the Developing Pancreas of a Neurogenin3/EGFP Transgenic Mouse.

Neurogenin 3 (ngn3) is a basic helix loop helix transcription factor that is transiently expressed in the developing mouse pancreas with peak expression around E15. In mice lacking the ngn3 gene the endocrine cells of the pancreas fail to develop suggesting that the ngn3-positive cell may represent a progenitor cell for the endocrine pancreas. In order to purify and characterize this cell in detail we have generated a transgenic mouse, in which the ngn3 promoter drives expression of enhanced green fluorescent protein (EGFP). In the E15.5 embryo EGFP was expressed in the dorsal and ventral pancreas, the duodenum, and lower intestine as well as in the brain. This pattern of expression was in keeping with the known expression profile of the endogenous ngn3 gene. Within the pancreas EGFP was localized in close proximity to cells that stained positive for ngn3, insulin, and glucagon, but was absent from regions of the pancreas that stained positive for amylase. EGFP was also present in the pancreas at E18.5, although there was no detectable expression of ngn3. At this stage EGFP did not colocalize with any of the hormones or exocrine markers. EGFP(+) cells were FACS purified (96%) from the E15 pancreas yielding approximately 10,000 cells or 1.6% of the total pancreatic cells from one litter. RT/PCR analysis confirmed that the purified cells expressed EGFP, ngn3, insulin, glucagon, somatostatin and pancreatic polypeptide. The ability to purify ngn3(+) cells provides an invaluable source of material for charactering in detail their properties.

Treatment of mice with 5-azacytidine efficiently activates silent retroviral genomes in different tissues.

The drug 5-azacytidine was injected into mice to activate silent retroviral genomes. The Mov-7 and Mov-10 substrains of mice were used, each of which carries a Moloney murine leukemia provirus with mutations in the coding regions at nonidentical positions. These proviral genomes are highly methylated and are not expressed in the animal. A single injection of the drug into postnatal mice induced transcription of the endogenous defective proviral genomes in thymus, spleen, and liver at 3 days after treatment. No viral transcription was detected in the brain of drug-exposed animals. When postnatal Mov-7/Mov-10 F1 mice were treated with the drug, infectious virus was generated efficiently and resulted in virus spread and viremia in all animals by 3 weeks of age. In contrast, infectious virus was not generated in F1 mice that had been treated during gestation with up to sublethal doses of the drug. Our results demonstrate that injection of 5-azacytidine can be used to efficiently and reproducibly activate silent genes in different cell populations of postnatal mice.