Genetically Engineered Swine Models to Study Diseases like Cystic Fibrosis

Scientifically Led National Enterprises PanelDate of publication unknownDate of publication unknownSwine have become important in biomedical research as they are excellent models for a variety of diseases including cardiovascular disease, atherosclerosis, cutaneous pharmacology, wound repair, cancer, diabetes, ophthalmology, toxicology research, lipoprotein metabolism, pathobiology of intestinal transport, injury and repair, as well as being considered potential sources of organs for xenotransplantation. Cystic Fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein is responsible for chloride ion transport. Disruption of the function in humans results in meconium ileus, focal biliary cirrhosis, pancreatic destruction, liver lesions, and lung disease. Unfortunately disruption of this gene in mice, while affecting chloride transport, does not result in any of the symptoms that humans develop. In collaboration with the University of Iowa we have created pigs with either a knockout of the CFTR gene or a mutated version that is similar to 70% of the people with CF (ΔF508). These pigs are born with meconium ileus, focal biliary cirrhosis, pancreatic destruction, liver lesions and they develop lung disease. Finally there is a model to invasively study the development of CF, to monitor disease progression, and to develop treatments and therapies without experimenting on children with CF

Creating genetically modified pigs by using nuclear transfer

Nuclear transfer (NT) is a procedure by which genetically identical individuals can be created. The technology of pig somatic NT, including in vitro maturation of oocytes, isolation and treatment of donor cells, artificial activation of reconstructed oocytes, embryo culture and embryo transfer, has been intensively studied in recent years, resulting in birth of cloned pigs in many labs. While it provides an efficient method for producing transgenic pigs, more importantly, it is the only way to produce gene-targeted pigs. So far pig cloning has been successfully used to produce transgenic pigs expressing the green fluorescence protein, expand transgenic pig groups and create gene targeted pigs which are deficient of alpha-1,3-galactosyltransferase. The production of pigs with genetic modification by NT is now in the transition from investigation to practical use. Although the efficiency of somatic cell NT in pig, when measured as development to term as a proportion of oocytes used, is not high, it is anticipated that the ability of making specific modifications to the swine genome will result in this technology having a large impact not only on medicine but also on agriculture

Cloning & Transgenic Swine for Medical and Agricultural Uses at MU

Comparative Medicine - OneHealth and Comparative Medicine Poster SessionThe pig is an important component of the world's food supply. As of December 1, 2009 the hog inventory in the United States of America was 65 million head. The United States is the world's third largest producer and second largest consumer, exporter, and importer of pork and pork products. Total farm income for hogs in 2008 has estimated to be $16.0 billion. Not only are pigs important to agriculture swine have become important in biomedical research as they are excellent models for cardiovascular disease, atherosclerosis, cutaneous pharmacology, wound repair, cancer, diabetes, ophthalmology, toxicology research, lipoprotein metabolism, pathobiology of intestinal transport, injury and repair, as well as being considered potential sources of organs for xenotransplantation. Furthermore, the swine genome is also quite similar to the human, as a phylogenetic approach using swine genome sequence data shows that the swine genome is 3x closer to the human than is the mouse. Reviewers at the NIH consider swine to be a very important model for human health and disease conditions as evidenced by the fact that for the past 6 years extramural support of research on swine has averaged over$115 million per year (NIH Office of the Director). The NIH considers the swine to be so important that it has helped establish the National Swine Resource and Research Center at the University of Missouri to serve as a genetic resource for the biomedical community

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility

Generation of cloned transgenic pigs rich in omega-3 fatty acids

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001). © 2006 Nature Publishing Group

A Genetic Porcine Model of Cancer

The large size of the pig and its similarity in anatomy, physiology, metabolism, and genetics to humans make it an ideal platform to develop a genetically defined, large animal model of cancer. To this end, we created a transgenic oncopig line encoding Cre recombinase inducible porcine transgenes encoding KRASG12D and TP53R167H, which represent a commonly mutated oncogene and tumor suppressor in human cancers, respectively. Treatment of cells derived from these oncopigs with the adenovirus encoding Cre (AdCre) led to KRASG12D and TP53R167H expression, which rendered the cells transformed in culture and tumorigenic when engrafted into immunocompromised mice. Finally, injection of AdCre directly into these oncopigs led to the rapid and reproducible tumor development of mesenchymal origin. Transgenic animals receiving AdGFP (green fluorescent protein) did not have any tumor mass formation or altered histopathology. This oncopig line could thus serve as a genetically malleable model for potentially a wide spectrum of cancers, while controlling for temporal or spatial genesis, which should prove invaluable to studies previously hampered by the lack of a large animal model of cancer

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock

Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk

Both the low animal cell density of bioreactors and their ability to post-translationally process recombinant factor IX (rFIX) limit hemophilia B therapy to transgenic pigs to make rFIX in milk at about 3,000-fold higher output than provided by industrial bioreactors. However, this resulted in incomplete γ-carboxylation and propeptide cleavage where both processes are transmembrane mediated. We then bioengineered the co-expression of truncated, soluble human furin (rFurin) with pro-rFIX at a favorable enzyme to substrate ratio. This resulted in the complete conversion of pro-rFIX to rFIX while yielding a normal lactation. Importantly, these high levels of propeptide processing by soluble rFurin did not preempt γ-carboxylation in the ER and therefore was compartmentalized to the Trans-Golgi Network (TGN) and also to milk. The Golgi specific engineering demonstrated here segues the ER targeted enhancement of γ-carboxylation needed to biomanufacture coagulation proteins like rFIX using transgenic livestock

Unraveling Twisty Linear Polarization Morphologies in Black Hole Images

We investigate general relativistic magnetohydrodynamic simulations (GRMHD) to determine the physical origin of the twisty patterns of linear polarization seen in spatially resolved black hole images and explain their morphological dependence on black hole spin. By characterising the observed emission with a simple analytic ring model, we find that the twisty morphology is determined by the magnetic field structure in the emitting region. Moreover, the dependence of this twisty pattern on spin can be attributed to changes in the magnetic field geometry that occur due to the frame dragging. By studying an analytic ring model, we find that the roles of Doppler boosting and lensing are subdominant. Faraday rotation may cause a systematic shift in the linear polarization pattern, but we find that its impact is subdominant for models with strong magnetic fields and modest ion-to-electron temperature ratios. Models with weaker magnetic fields are much more strongly affected by Faraday rotation and have more complicated emission geometries than can be captured by a ring model. However, these models are currently disfavoured by the recent EHT observations of M87*. Our results suggest that linear polarization maps can provide a probe of the underlying magnetic field structure around a black hole, which may then be usable to indirectly infer black hole spins. The generality of these results should be tested with alternative codes, initial conditions, and plasma physics prescriptions.Comment: 25 pages, 19 figure