Jeffrey J. Whyte

Vascular Effects of Exercise: Endothelial Adaptations Beyond Active Muscle Beds

Endothelial adaptations to exercise training are not exclusively conferred within the active muscle beds. Herein, we summarize key studies that have evaluated the impact of chronic exercise on the endothelium of vasculatures perfusing nonworking skeletal muscle, brain, viscera, and skin, concluding with discussion of potential mechanisms driving these endothelial adaptations.

Genetic Modifications of Pigs for Medicine and Agriculture

Genetically modified swine hold great promise in the fields of agriculture and medicine. Currently, these swine are being used to optimize production of quality meat, to improve our understanding of the biology of disease resistance, and to reduced waste. In the field of biomedicine, swine are anatomically and physiologically analogous to humans. Alterations of key swine genes in disease pathways provide model animals to improve our understanding of the causes and potential treatments of many human genetic disorders. The completed sequencing of the swine genome will significantly enhance the specificity of genetic modifications, and allow for more accurate representations of human disease based on syntenic genes between the two species. Improvements in both methods of gene alteration and efficiency of model animal production are key to enabling routine use of these swine models in medicine and agriculture. Mol. Reprod. Dev. 78:879–891, 2011.

Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs

We report using zinc finger nucleases (ZFNs) and somatic cell nuclear transfer (SCNT) to generate pigs with a knockout (KO) mutation of an enhanced green fluorescent protein (eGFP) transgene. ZFNs are synthetic modular proteins composed of a FokI endonuclease domain linked to a sequence-specific zinc finger DNA-binding domain. Pairs of ZFNs bind to the target region, allowing FokI dimerization and subsequent DNA cleavage. Mutation of an eGFP transgene by using ZFNs has been demonstrated in rats (Geurts et al., 2009); however, there are no reports of ZFN-mutated livestock offspring. Using ZFNs to increase the efficiency of gene modification may advance the production of agriculturally and clinically relevant animal models, particularly in species where modification is difficult. All animal procedures followed an approved IACUC protocol. Adult porcine ear fibroblasts hemizygous for the eGFP transgene (Whitworth et al., 2009), from a grandson of the founder animal, were cultured to 75% confluency, trypsinized, and cotransfected (Ross et al., 2010) with a pair of ZFN plasmids that bind to opposing strands at the eGFP target site (Sigma–Aldrich CompoZr®) and a red fluorescent CAG-tomato plasmid as a transient selectable fluorophore reporting transfection efficiency. Transfected fibroblasts were cultured for 96 h and selected for red fluorescence by automated cell sorting (FACS; Fig. 1A,B) and seeded into 96-well plates (100 cells/well) based on CAG-tomato expression (2% of total cells sorted). A second round of FACS using negative selection of eGFP fluorescence enriched for eGFP KO cells (~5% of sorted cells). PCR of genomic DNA from fibroblasts in wells containing predominantly non-green cells was used to amplify a fragment bracketing the ZFN targeting site. The PCR product was cloned into E. coli, and 16 colonies showed mutations by Sanger sequencing. These mutations at the ZFN cleavage site included a 6-bp deletion, a 333-bp deletion, and a 222-bp deletion replaced with a 113-bp inversion of the deleted sequence (Supplemental Information http://animalsciences.missouri.edu/faculty/prather/). Fibroblast colonies determined to carry ZFN-induced mutations were used as donor cells for SCNT, and embryo transfer (n = 3; Whitworth et al., 2009). One Day-12 embryo collection recovered 7 of 9 embryos that did not fluoresce. Quality sequence was obtained from four embryos, and confirmed that the two embryos that fluoresced had an intact eGFP and two embryos that did not fluoresce were mutated. One of seven piglets recovered via Cesarean section at term for the other two pregnancies fluoresced (Fig. 1C). DNA sequencing confirmed that this piglet had unaltered sequence at the predicted ZFN cut site, while the non-fluorescing piglets had various deletions and insertions. While a direct comparison to a 12% mutation rate in the rat made by microinjection (Geurts et al., 2009) cannot be made because of our FACS preselection and SCNT methods, six out of seven founders being mutated is consistent with their results. This study establishes ZFN-based gene modification in a large animal model. The methods used are anticipated to be useful in selective knockout of endogenous swine genes of agricultural and biomedical importance without introducing any transgenic sequence into the genome. Figure 1 A: Control porcine ear fibroblasts; eGFP and Hoechst DNA stain fluorescence. B: Porcine ear fibroblasts treated with eGFP-specific ZFNs; eGFP and Hoechst DNA stain fluorescence. GFP fluorescence is absent in a high proportion of the treated fibroblasts ...

Genetically Engineered Pig Models for Human Diseases

Although pigs are used widely as models of human disease, their utility as models has been enhanced by genetic engineering. Initially, transgenes were added randomly to the genome, but with the application of homologous recombination, zinc finger nucleases, and transcription activator-like effector nuclease (TALEN) technologies, now most any genetic change that can be envisioned can be completed. To date these genetic modifications have resulted in animals that have the potential to provide new insights into human diseases for which a good animal model did not exist previously. These new animal models should provide the preclinical data for treatments that are developed for diseases such as Alzheimers disease, cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy, diabetes, and organ failure. These new models will help to uncover aspects and treatments of these diseases that were otherwise unattainable. The focus of this review is to describe genetically engineered pigs that have resulted in models of human diseases.

The effects of acute and chronic exercise on the vasculature

Regular physical activity (endurance training, ET) has a strong positive link with cardiovascular health. The aim of this review is to draw together the current knowledge on gene expression in different cell types comprising the vessels of the circulatory system, with special emphasis on the endothelium, and how these gene products interact to influence vascular health. The effect beneficial effects of ET on the endothelium are believed to result from increased vascular shear stress during ET bouts. A number of mechanosensory mechanisms have been elucidated that may contribute to the effects of ET on vascular function, but there are questions regarding interactions among molecular pathways. For instance, increases in flow brought on by ET can reduce circulating levels of viscosity and haemostatic and inflammatory variables that may interact with increased shear stress, releasing vasoactive substances such as nitric oxide and prostacyclin, decreasing permeability to plasma lipoproteins as well as the adhesion of leucocytes. At this time the optimal rate‐of‐flow and rate‐of‐change in flow for determining whether anti‐atherogenic or pro‐atherogenic processes proceed remain unknown. In addition, the impact of haemodynamic variables differs with vessel size and tissue type in which arteries are located. While the hurdles to understanding the mechanism responsible for ET‐induced alterations in vascular cell gene expression are significant, they in no way undermine the established benefits of regular physical activity to the cardiovascular system and to general overall health. This review summarizes current understanding of control of vascular cell gene expression by exercise and how these processes lead to improved cardiovascular health.

Effect of epigenetic regulation during swine embryogenesis and on cloning by nuclear transfer

Swine play important roles as models of human disease. A combination of genetic modification with somatic cell nuclear transfer (SCNT) holds the promise of uncovering the pathogenesis of human diseases and then of developing therapeutic protocols. Unfortunately, the mechanism(s) of nuclear remodeling (a change in the structure of the nucleus) and reprogramming (a change in the transcriptional profile) during SCNT remains unclear. Incomplete remodeling is thought to cause lower cloning efficiency and abnormalities in cloned embryos and offspring. Here, we review the epigenetic regulatory and remodeling events that occur during preimplantation development of embryos derived from fertilization or SCNT, with a focus on DNA methylation and histone modifications. The discussion ends with a description of attempts at assisted remodeling of the donor somatic cell nucleus and the SCNT embryo.

Method of Oocyte Activation Affects Cloning Efficiency in Pigs

The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken β‐actin/rabbit β‐globin hybrid promoter (pCAGG‐EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca2+ followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst‐stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG‐EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. Mol. Reprod. Dev. 76: 490–500, 2009.

Effects of Diets Enriched in Omega-3 and Omega-6 Polyunsaturated Fatty Acids on Offspring Sex-Ratio and Maternal Behavior in Mice

Abstract There have been many trials describing the effects of polyunsaturated fatty acids (PUFA) on fecundity, neonatal development, and maternal behavior in humans, but few controlled studies in rodents. We examined the effects of a maternal diet high in omega 3 (N-3) or omega 6 (N-6) PUFA on NIH Swiss mice. Female mice were ad libitum fed one of three complete and balanced diets (N-3, enriched in menhaden oil; N-6, enriched in corn oil; C, control diet, Purina 5015) from age 4 wk until the end of the study. Mice were bred at ∼19 wk and 27 wk of age, providing a total of 838 pups from 129 litters in two experiments. After weaning their pups from parity 1, behavior of dams was assessed on elevated-plus and open-field mazes. Although the fraction of male pups from the N-3 and C groups was not different from 0.5, dams on the N-6 diet birthed more daughters than sons (213 vs. 133; P < 0.001). Although maternal stress has been reported to favor birth of daughters, the behavior of N-6 dams was not different from controls. By contrast, the N-3 dams displayed greater anxiety, spending less time in the open arms and more time in the closed arms of the elevated maze and traveling less distance and exhibiting less exploratory behavior in the open field (P < 0.05). N-3 dams tended to produce smaller litters than C dams, and N-3-suckled pups gained less weight (P < 0.05). In conclusion, the N-3 diet had negative effects on murine fecundity and maternal behavior, whereas the N-6 diet favored birth of daughters.

Tracing the Stemness of Porcine Skin-Derived Progenitors (pSKP) Back to Specific Marker Gene Expression

Multipotent skin-derived progenitors (SKP) can produce both neural and mesodermal progeny in vitro, sharing the characteristics of embryonic neural crest stem cells. However, the molecular basis for the property of multiple lineage potential and neural crest origin of SKPs is still elusive. Here we report the cooperative expression of pluripotency related genes (POU5F1, SOX2, NANOG, STAT3) and neural crest marker genes (p75NTR, TWIST1, PAX3, SNAI2, SOX9, SOX10) in GFP-transgenic porcine skin-derived progenitors (pSKP). The proportion of cells positive for POU5F1, nestin, fibronectin, and vimentin were 12.3%, 15.1%, 67.9% and 53.7%, showing the heterogeneity of pSKP spheres. Moreover, pSKP cells can generate both neural (neurons and glia) and mesodermal cell types (smooth muscle cells and adipocytes) in vitro, indicating the multiple lineage potency. Four transcription factors (POU5F1, SNAI2, SOX9, and PAX3) were identified that were sensitive to mitogen (FBS) and/or growth factors (EGF and bFGF). We infer that POU5F1, SNAI2, SOX9, and PAX3 may be the key players for maintaining the neural crest derived multipotency of SKP cells in vitro. This study has provided new insight into the molecular mechanism of stemness for somatic-derived stem cells at the level of transcriptional regulation.

The Contrasting Effects of Ad Libitum and Restricted Feeding of a Diet Very High in Saturated Fats on Sex Ratio and Metabolic Hormones in Mice

Abstract Skewing of the sex ratio towards males occurs among pups born to mice fed a very high saturated fat (VHF) diet. In the present study, we tested whether the fat content of the VHF diet rather than the number of calories consumed is responsible for this effect. Eight-week-old NIH Swiss mice were placed on the VHF diet either ad libitum (VHF) or in a restricted manner (VHF-R). The VHF-R mice gained weight at a similar rate to controls fed a standard chow diet. Mice were bred at 15 wk and subsequently at 26 wk and 35 wk of age. Overall, the VHF, VHF-R, and control groups delivered 244, 242, and 274 pups, respectively, with male proportions of 0.60, 0.43, and 0.48, respectively. The pup sex ratios of the VHF group (favoring males) and VHF-R group (favoring females) each differed from 0.5 (P < 0.01). The sex ratios also differed (P < 0.0001) between the VHF and control groups, and between the VHF and VHF-R groups. Within the diet groups, maternal body weight had no effect on sex ratio. Serum leptin concentrations among the dams were similar in the VHF and VHF-R groups but higher than in the control group, while the IGF1 and corticosterone levels were comparable in all three groups. Therefore, the atypical sex ratios of offspring born to dams on the VHF diet seem to be influenced by the amount of fat consumed. Since males fed the VHF diet had neither more Y-sperm nor sired more sons than daughters, the dietary effects are manifested exclusively through the female.