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

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

A mixture of thymol and carvacrol mitigates inflammation and oxidative stress responses in intestinal epithelial cell models

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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.</p

Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling

Duchenne muscular dystrophy is typically diagnosed in the preschool years because of locomotor defects, indicative of muscle damage. Thus, effective therapies must be able to rescue muscle from further decline. We have established that Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) gene transfer will prevent many aspects of dystrophic pathology, likely through up-regulation of utrophin and increased oxidative capacity, however, the extent to which it will rescue muscle with disease manifestations has not been determined. Our hypothesis is that gene transfer of PGC-1α into declining muscle will reduce muscle injury compared to control muscle. To test our hypothesis AAV6 driving expression of PGC-1α was injected into single hind limbs of 3 week old mdx mice while the contralateral limb was given a sham injection. At six weeks of age treated solei had 37% less muscle injury compared to sham-treated muscles (pThis is a manuscript of an article published as Hollinger, Katrin, Delphine Gardan-Salmon, Connie Santana, Drance Rice, Elizabeth Snella, and Joshua T. Selsby. "Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 305, no. 1 (2013): R13-R23. doi: 10.1152/ajpregu.00221.2012. Posted with permission.</p

Enriching sow environment and diet during gestation reduced piglet neonatal mortality

Sow environment during gestation can generate maternal stress which could influence piglet health and survival after birth. The study aimed to investigate a strategy of environmental and nutritional enrichment to reduce maternal stress and its consequences on piglet mortality. Gestating sows were group-housed in a conventional system on a slatted floor (C, n=26), in the same conventional system with environmental and nutritional enrichment (CE, n=30) or in larger pens enriched with straw bedding (E, n=27). The enrichment of the CE group consisted of pieces of oak attached to a chain (three per pen) and straw pellets provided in the trough at a rate of 200 g/d from 3-30 days of gestation (DG) and 400 g/d from 31-104 DG. On DG 105, sows were transferred into farrowing pens and housed in identical individual stalls on a slatted floor. Cortisol concentration was measured in sow saliva during gestation, sow behavioural and investigative activities were recorded on DG 101 and piglet mortality was recorded. Cortisol concentration was greater (P<0.05) in C and CE than in E sows on DG 14 while it was intermediate in CE sows compared with C and E sows on DG 105 (before transfer to farrowing pens), and no longer differed among the 3 treatments on DG 107 (after the transfer). On DG 101, CE sows exhibited a lower proportion of stereotypies compared to C sows (22 vs 34%, P<0.05) but a greater proportion compared to E sows (7%, P<0.05). On this same day, CE sows had more investigative sequences than C sows (7.3±7.0 vs 1.7±1.8, P<0.01) but less than E sows (20.3±13.8; P<0.01). Rate of early mortality (i.e. piglets dead at birth + piglets that died within 12 h of birth) was lower in groups CE and E (6.6 and 6.3%, respectively) than in group C (11.1%, P<0.05), but overall mortality (stillbirth + preweaning death) did not differ significantly among the 3 groups (23.2, 19.1 and 19.3% in groups C, CE, and E, respectively, P=0.35). Enriching the sow environment and diet during gestation therefore improved sow welfare and reduced piglet mortality at and soon after birth. Research was funded by the EU FP7 Prohealth project (no. 613574)

Housing systems influence gut microbiota composition of sows but not of their piglets

Different housing systems can be used in pig production and little is known about their effect on gut microbiota composition. In this study we characterized fecal microbiota by sequencing the rRNA genes in sows kept during gestation in conventional pens with a slatted floor and in enriched pens with a floor covered with deep straw. After farrowing, microbiota of 1- and 4-day-old piglets were also monitored. Microbiota of sows from the enriched system contained significantly more Prevotella, Parabacteroides, CF231, Phascolarctobacterium, Fibrobacter, Anaerovibrio and YRC22 and significantly less Lactobacillus, Bulleidia, Lachnospira, Dorea, Ruminococcus and Oscillospira than microbiota of sows from the conventional system. The Firmicutes to Bacteroidetes ratio was 0.96 in the microbiota of sows kept in the enriched pens and this increased to 1.66 in the microbiota of sows kept in the conventional system. The production system therefore influenced microbiota composition, most likely due the ingestion of the straw. The microbiota of 1- and 4-day-old piglets differed from the microbiota of sows and sows therefore did not represent the most important source for their colonization in early days of life

Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling

Duchenne muscular dystrophy is typically diagnosed in the preschool years because of locomotor defects, indicative of muscle damage. Thus, effective therapies must be able to rescue muscle from further decline. We have established that Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) gene transfer will prevent many aspects of dystrophic pathology, likely through up-regulation of utrophin and increased oxidative capacity, however, the extent to which it will rescue muscle with disease manifestations has not been determined. Our hypothesis is that gene transfer of PGC-1α into declining muscle will reduce muscle injury compared to control muscle. To test our hypothesis AAV6 driving expression of PGC-1α was injected into single hind limbs of 3 week old mdx mice while the contralateral limb was given a sham injection. At six weeks of age treated solei had 37% less muscle injury compared to sham-treated muscles (p\u3c0.05). Resistance to contraction induced injury was improved 10% (p\u3c0.05) likely driven by the 5-fold (p\u3c0.05) increase in utrophin protein expression and increase in dystrophin associated complex members. Treated muscles were more resistant to fatigue, which was likely caused by the corresponding increase in oxidative markers. PGC-1α over-expressing limbs also exhibited increased expression of genes related to muscle repair and autophagy. These data indicate that the PGC-1α pathway remains a good therapeutic target as it reduced muscle injury and improved function using a rescue paradigm. Further, these data also indicate that the beneficial effects of PGC-1α gene transfer are more complex than increased utrophin expression and oxidative gene expression