Utility and Efficiency of Homologous Recombination for Introducing Targeted Modifications to the Pig Genome

The production and utilization of genetically modified animals greatly improves their utility in agriculture, as biomedical research models of human disease, for the production of recombinant pharmaceutical proteins, and for production of organs with greater potential for xenotransplantation. While numerous strategies have been utilized in the production of transgenic large animals, cell-based transgenesis followed by somatic cell nuclear transfer (SCNT) is currently the most widely applied method. We constructed a targeting vector with 2774 and 1890 bp arms of homology flanking a neomycin resistant gene. Following delivery of the DNA targeting vector into the porcine fetal fibroblasts by electroporation and selection with G418, PCR and sequencing confirmed one of 547 transgenic clonal colonies contained the targeted introduction of the neomycin resistance gene. Genetically modified fibroblast donor cells can be utilized for SCNT for the development of genetically modified pigs to study biological mechanisms important for animal agriculture and to create biomedical models

Effects of In-utero Heat Stress on Porcine Post-natal Thermoregulation

Pigs were exposed to heat stress (HS) and thermoneutral (TN) conditions in-utero. Post-natally, they were exposed to either TN or HS environments for 15 days, and differences in physiological response comparing inutero treatment groups were determined. Our results indicated that gestational HS (GHS) pigs had increased core body temperature during post-natal HS compared to pigs exposed to gestational TN (GTN); however, there were no production differences between gestational groups. This provides evidence suggesting pigs exposed to in-utero HS may have an increased tolerance to post-natal HS, at least from a productivity standpoint

Plethysmography measurements of respiratory function in conscious unrestrained mice

Whole body plethysmography (WBP) is used to quantify pulmonary function in conscious, unrestrained mice. We determined currently whether time of day and environmental lighting influence day-to-day reproducibility of pulmonary function, and quantifed the necessary habituation time in the WBP chamber. Two-month-old male C57BL6 and mdx mice (n = 8/group, reverse light cycle), were examined on consecutive days using a calibrated WBP chamber and manufacturer software was used to calculate respiratory measures. Respiratory data stabilized between 5–10 min for all variables. Mice exhibited time of day respiratory differences, performing more forceful and less frequent breaths midday (11:45 a.m. and 3:00 p.m.) compared to 7:30 a.m. WBP performed in darkened conditions elicited more forceful breathing than lit conditions. Day-to-day reproducibility during controlled conditions ranged from r 2 = 0.58 to 0.62 for the functional measures. Findings indicate reproducible respiratory data are obtainable following a 15-min chamber habituation and standardization of time of day and room lighting

PGC-1α overexpression increases transcription factor EB nuclear localization and lysosome abundance in dystrophin-deficient skeletal muscle

Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin protein and results in progressive muscle wasting. Dystrophin deficiency leads to a host of dysfunctional cellular processes including impaired autophagy. Autophagic dysfunction appears to be due, at least in part, to decreased lysosomal abundance mediated by decreased nuclear localization of transcription factor EB (TFEB), a transcription factor responsible for lysosomal biogenesis. PGC‐1α overexpression decreased disease severity in dystrophin‐deficient skeletal muscle and increased PGC‐1α has been linked to TFEB activation in healthy muscle. The purpose of this study was to determine the extent to which PGC‐1α overexpression increased nuclear TFEB localization, increased lysosome abundance, and increased autophagosome degradation. We hypothesized that overexpression of PGC‐1α would drive TFEB nuclear translocation, increase lysosome biogenesis, and improve autophagosome degradation. To address this hypothesis, we delivered PGC‐1α via adeno‐associated virus (AAV) vector injected into the right limb of 3‐week‐old mdx mice and the contralateral limbs received a sham injection. At 6 weeks of age, this approach increased PGC‐1α transcript by 60‐fold and increased TFEB nuclear localization in gastrocnemii from PGC‐1α treated limbs by twofold compared to contralateral controls. Furthermore, lamp2, a marker of lysosome abundance, was significantly elevated in muscles from limbs overexpressing PGC‐1α. Lastly, increased LC3II and similar p62 in PGC‐1α overexpressing‐limbs compared to contralateral limbs are supportive of increased degradation of autophagosomes. These data provide mechanistic insight into PGC‐1α‐mediated benefits to dystrophin‐deficient muscle, such that increased TFEB nuclear localization in dystrophin‐deficient muscle leads to increased lysosome biogenesis and autophagy

Why we should sweat heat stress

Core body temperature of pigs increases when the rate of heat accumulation exceeds the rate ofheat loss. This increase in core body temperature removes the animal from a thermal comfort zonecausing heat stress. The acute, severe effects of heat stress on humans can result in death and aregenerally well-reported in the news (e.g. European heat wave of 2003, Middle East- 2015, France-2019)

Nutraceutical and pharmaceutical cocktails did not improve muscle function or reduce histological damage in D2-mdx mice

Progressive muscle injury and weakness are hallmarks of Duchenne muscular dystrophy. We showed previously that quercetin (Q) partially protected dystrophic limb muscles from disease-related injury. As quercetin activates PGC-1α through Sirtuin-1, an NAD+-dependent deacetylase, the depleted NAD+ in dystrophic skeletal muscle may limit quercetin efficacy, hence, supplementation with the NAD+ donor, nicotinamide riboside (NR), may facilitate quercetin efficacy. Lisinopril (Lis) protects skeletal muscle and improves cardiac function in dystrophin-deficient mice, therefore it was included in this study to evaluate the effects of lisinopril used with quercetin and NR. Our purpose was to determine the extent to which Q, NR, and Lis decreased dystrophic injury. We hypothesized that Q, NR or Lis alone would improve muscle function and decrease histological injury and when used in combination would have additive effects. Muscle function of 11-month-old DBA (healthy), D2-mdx (dystrophin-deficient), and D2-mdx mice was assessed following treatment with Q, NR, and/or Lis for 7-months. To mimic typical pharmacology of DMD patients a group was treated with prednisolone (Pred) in combination with Q, NR and Lis. At 11-months of age, dystrophin deficiency decreased specific tension and tetanic force in the soleus and extensor digitorum longus muscles and was not corrected by any treatment. Dystrophic muscle was more sensitive to contraction-induced injury, which was partially offset in the QNRLisPred group, while fatigue was similar between all groups. Treatments did not decrease histological damage. These data suggest treatment with Q, NR, Lis and Pred failed to adequately maintain dystrophic limb muscle function or decrease histological 46 damage

Dystrophin insufficiency causes selective muscle injury and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle

The purpose of this investigation was to determine the extent to which dystrophin insufficiency caused histomorphological changes in a novel pig model of Becker muscular dystrophy. In our procedures, we used a combination of biochemical approaches, including quantitative PCR and Western blots, along with a histological analysis using standard and immunohistological measures. We found that 8‐wk‐old male affected pigs had a 70% reduction in dystrophin protein abundance in the diaphragm, psoas major, and longissimus lumborum and a 5‐fold increase in serum creatine kinase activity compared with healthy male littermates. Dystrophin insufficiency in the diaphragm and the longissimus resulted in muscle histopathology with disorganized fibrosis that often colocalized with fatty infiltration but not the psoas. Affected animals also had an 80–85% reduction in α‐sarcoglycan localization in these muscles, indicating compromised assembly of the dystrophin glycoprotein complex. Controls used in this study were 4 healthy male littermates, as they are most closely related to the affected animals. We concluded that pigs with insufficient dystrophin protein expression have a phenotype consistent with human dystrophinopathy patients. Given that and their similarity in body size and physiology to humans, we further conclude that this pig line is an appropriate translational model for dystrophinopathies.—Hollinger, K., Yang, C. X., Montz, R. E., Nonneman, D., Ross, J. W., Selsby, J. T. Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin‐glycoprotein complex assembly in pig skeletal muscle

Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models

Background: Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. Methods: Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. Results: Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. Conclusion: Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction

Heat stress causes oxidative stress but not inflammatory signaling in porcine skeletal muscle

Heat stress is associated with death and other maladaptions including muscle dysfunction and impaired growth across species. Despite this common observation, the molecular effects leading to these pathologic changes remain unclear. The purpose of this study was to determine the extent to which heat stress disrupted redox balance and initiated an inflammatory response in oxidative and glycolytic skeletal muscle. Female pigs (5–6/group) were subjected to thermoneutral (20 °C) or heat stress (35 °C) conditions for 1 or 3 days and the semitendinosus removed and dissected into red (STR) and white (STW) portions. After 1 day of heat stress, relative abundance of proteins modified by malondialdehyde, a measure of oxidative damage, was increased 2.5-fold (P \u3c 0.05) compared with thermoneutral in the STR but not the STW, before returning to thermoneutral conditions following 3 days of heat stress. This corresponded with increased catalase and superoxide dismutase-1 gene expression (P \u3c 0.05) and superoxide dismutase-1 protein abundance (P \u3c 0.05) in the STR but not the STW. In the STR catalase and total superoxide dismutase activity were increased by ~30% and ~130%, respectively (P \u3c 0.05), after 1 day of heat stress and returned to thermoneutral levels by day 3. One or 3 days of heat stress did not increase inflammatory signaling through the NF-κB pathway in the STR or STW. These data suggest that oxidative muscle is more susceptible to heat stress-mediated changes in redox balance than glycolytic muscle during chronic heat stress