Cell Biology Symposium: Zinc finger nucleases to create custom-designed modifications in the swine (Sus scrofa) genome.

Engineered zinc finger nucleases (ZFN) are rapidly gaining popularity as a means to enhance the rate and specificity of DNA modifications in plant and animal cells. Repair-mediated gene modification by ZFN is driven by introducing DNA double-strand breaks via a nonspecific nuclease domain linked to a sequence-specific zinc finger nucleotide recognition domain. This review examines the use of ZFN to produce genetically modified swine and the potential of this technology for the future. By combining conventional gene targeting methods with somatic cell nuclear transfer, several genetically modified pig models have been produced. These conventional techniques are inefficient in mammalian somatic cells and provide little control over the site specificity and rate of exogenous DNA integration. The use of engineered ZFN that bind and cleave genomic DNA at specific loci can enhance targeting efficiencies by orders of magnitude. Recent publication of the first genetic modification in pigs by combining ZFN technology with somatic cell nuclear transfer has opened the door to genome targeting with a precision that was not previously possible in a large animal model. Since that time, model pigs with selective knockout of endogenous genes have been produced. This review will examine the use of ZFN to generate these pig models and the potential of ZFN to accelerate the production of genetically modified pigs of agricultural and biomedical importance. Current methods of ZFN design, important considerations for their safe and effective use in modification of the swine genome, and future innovative applications of this technology in pigs will be discussed.

Vascular endothelium-specific overexpression of human catalase in cloned pigs.

The objective of this study was to develop transgenic Yucatan minipigs that overexpress human catalase (hCat) in an endothelial-specific manner. Catalase metabolizes hydrogen peroxide (H(2)O(2)), an important regulator of vascular tone that contributes to diseases such as atherosclerosis and preeclampsia. A large animal model to study reduced endothelium-derived H(2)O(2) would therefore generate valuable translational data on vascular regulation in health and disease. Yucatan minipig fetal fibroblasts stably co-transfected with human catalase (Tie2-hCat) and eGFP expression constructs were isolated into single-cell populations. The presence of the Tie2-hCat transgene in individual colonies of fibroblasts was determined by PCR. Transgenic fibroblasts were used for nuclear transfer into enucleated oocytes by electrofusion. A minimum of 140 cloned embryos were transferred per surrogate sow (n = 4). All four surrogates maintained pregnancies and piglets were delivered by cesarean section. Nine male piglets from three of the four litters carried the Tie2-hCat transgene. Expression of human catalase mRNA and overall elevated catalase protein in isolated umbilical endothelial cells from transgenic piglets were verified by RT-PCR and western blot, respectively, and endothelial localization was confirmed by immunohistochemistry. Increased enzymatic activity of catalase in transgenic versus wild-type endothelial cells was inferred based on significantly reduced levels of H(2)O(2) in culture. The similarities in swine and human cardiovascular anatomy and physiology will make this pig model a valuable source of information on the putative role of endothelium-derived H(2)O(2) in vasodilation and in the mechanisms underlying vascular health and disease.

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.

Fluorescent in situ hybridization for sex chromosome determination before and after fertilization in mice.

In mice, the relative numbers of male and female pups per litter not only can vary but can probably change over the course of pregnancy in response to numerous environmental and physiological factors. As such, a technique is required to determine gender at several developmental stages. Here we describe a robust and accurate fluorescent in situ hybridization (FISH) procedure for determining chromosomal sex that can be applied with minimal modification to sperm, pre-and post-implantation conceptuses and recovered dead post-natal pups. Sperm was prepared for FISH analysis y using a modified microwave decondensation-denaturation technique. Preimplantation conceptuses (0.5dpc) were cultured to the morula stage before sexing. They were then acid-treated to remove the zona pellucida. Tissue homogenates from postimplantational conceptuses (8.5dpc) and stillborn pups were fixed to pre-etched slides. Specimens were hybridized with identical, commercially available DNA probes for the X (FITC) and Y (Cy3) chromosomes. Sperm ratios met the expected value of 0.5 when determined by using XY FISH. Preimplantation conceptuses pre-treated with pepsin yielded distinct fluorescence of X and Y chromosomes in morulae, whereas microwave decondensation resulted in loss of conceptuses from the slide. Both 4.0 and 8.5dpc conceptuses displayed mean sex ratios of 0.5. Post-natal FISH analysis allowed gender identification of pups that could not be sexed due to developmental abnormalities or partial cannibalism. FISH analysis of sperm and of multiple conceptuses or post-natal tissue provided a cost-effective, accurate alternative to PCR-based sex determination.

Maternal diet composition alters serum steroid and free fatty acid concentrations and vaginal pH in mice.

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17beta-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 +/- 3.0 pg/ml), compared with LF mice (5.2 +/- 2.3 pg/ml; P< or = 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 +/- 3.0 pg/ml) versus the LF group (32.7 +/- 8.4 pg/ml; P< or = 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 +/- 6.7 ng/ml) versus the 5015 group (60.1 +/- 4.9 ng/ml; P< or = 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 +/- 0.2 mmol/l) than LF and control mice (0.5 +/- 0.1 and 0.6 +/- 0.1 mmol/l respectively; P< or = 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 +/- 0.1 mmol/l) than LF and control females (0.4 +/- 0.1 and 0.6 +/- 0.1 mmol/l; P< or = 0.05). Mean vaginal pH of VHF females (6.41 +/- 0.09) was lower than 5015 females (6.76 +/- 0.10; P< or = 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

The effect of creosote on vitellogenin production in rainbow trout (Oncorhynchus mykiss).

As part of a broader investigation into the effects of creosote treatments on the aquatic biota in pond microcosms, we examined the possible implications for vitellogenin (Vtg) production in Oncorhynchus mykiss [rainbow trout (RT)]. Vtg is the precursor of egg yolk protein and has emerged as a useful biomarker of exposure to estrogenic substances. Our a priori intent was to assess the ability of the creosote treatments (nominal cresoste concentrations were 0, 3, and 10 microl/L immediately after the last subsurface addition) to induce estrogenic responses in RT. The data showed no evidence of an estrogenic response in the treated fish. During the course of the experiment, however, the fish matured and began to produce Vtg, probably in response to endogenous estrogen. A posteriori analysis of the Vtg data from the maturing fish showed that after 28 days, the plasma Vtg concentrations were about 15-fold lower in fish from the creosote-treated microcosms compared with fish from the reference microcosm. Although the experiment design does not permit mechanistic insights, our observation suggests that exposure of female fish to PAH mixtures such as creosote can impair the production of Vtg with possible health implications for embryos and larvae.

The H4IIE cell bioassay as an indicator of dioxin-like chemicals in wildlife and the environment.

The H4IIE cell bioassay has proven utility as a screening tool for planar halogenated hydrocarbons (PHHs) and structurally similar chemicals accumulated in organisms from the wild. This bioassay has additional applications in hazard assessment of PHH exposed populations. In this review, the toxicological principles, current protocols, performance criteria, and field applications for the assay are described. The H4IIE cell bioassay has several advantages over the analytical measurement of PHHs in environmental samples, but conclusions from studies can be strengthened when both bioassay and analytical chemistry data are presented together. Often, the bioassay results concur with biological effects in organisms and support direct measures of PHHs. For biomonitoring purposes and prioritization of PHH-contaminated environments, the H4IIE bioassay may be faster and less expensive than analytical measurements. The H4IIE cell bioassay can be used in combination with other biomarkers such as in vivo measurements of CYP1A1 induction to help pinpoint the sources and identities of dioxin-like chemicals. The number of studies that measure H4IIE-derived TCDD-EQs continues to increase, resulting in subtle improvements over time. Further experiments are required to determine if TCDD-EQs derived from mammalian cells are adequate predictors of toxicity to non-mammalian species. The H4IIE cell bioassay has been used in over 300 published studies, and its combination of speed, simplicity, and ability to integrate the effects of complex contaminant mixtures makes it a valuable addition to hazard assessment and biomonitoring studies.

Effects of creosote exposure on rainbow trout pronephros phagocyte activity and the percentage of lymphoid B cells.

An outdoor microcosm study was conducted in order to evaluate the kinetics of immunomodulation in rainbow trout (Oncorhynchus mykiss) exposed to liquid creosote. Fish were sampled on d 7, 14, 21, and 28 from microcosms dosed initially with 0, 3, and 10 microl/L of creosote. Pronephros leukocytes were monitored for phagocytic activity, oxidative burst, and surface immunoglobulin-positive (Slg+) B-cell counts. Oxidative burst was inhibited by creosote exposure; however, by sampling d 28, the burst response returned to near control levels. Phagocytic activity, on the other hand, was stimulated, peaking on sampling d 7, then returning to near control levels by d 28. Although control Slg+ B-cell counts were quite variable across sampling days, Slg+ B-cell counts were also elevated in creosote-exposed fish after seven days of exposure. Slg+ B-cell numbers decreased significantly to near control levels during the remainder of the study. The overall results from this study confirm that creosote has the potential to alter certain immune parameters, and emphasize the importance of monitoring changes in the immune system during exposure. Polycyclic aromatic hydrocarbons (PAHs), a major constituent of liquid creosote, are the suspected immune-altering agents.

Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure.

This review compiles and evaluates existing scientific information on the use, limitations, and procedural considerations for EROD activity (a catalytic measurement of cytochrome P4501A induction) as a biomarker in fish. A multitude of chemicals induce EROD activity in a variety of fish species, the most potent inducers being structural analogs of 2,3,7,8-tetracholordibenzo-p-dioxin. Although certain chemicals may inhibit EROD induction/activity, this interference is generally not a drawback to the use of EROD induction as a biomarker. The various methods of EROD analysis currently in use yield comparable results, particularly when data are expressed as relative rates of EROD activity. EROD induction in fish is well characterized, the most important modifying factors being fish species, reproductive status and age, all of which can be controlled through proper study design. Good candidate species for biomonitoring should have a wide range between basal and induced EROD activity (e.g., common carp, channel catfish, and mummichog). EROD activity has proven value as a biomarker in a number of field investigations of bleached kraft mill and industrial effluents, contaminated sediments, and chemical spills. Research on mechanisms of CYP1A-induced toxicity suggests that EROD activity may not only indicate chemical exposure, but also may also precede effects at various levels of biological organization. A current research need is the development of chemical exposure-response relationships for EROD activity in fish. In addition, routine reporting in the literature of EROD activity in standard positive and negative control material will enhance confidence in comparing results from different studies using this biomarker.

Effect of maintaining rainbow trout in creosote microcosms on lens optical properties and liver 7-ethoxyresorufin-O-deethylase (EROD) activity.

Previously, exposure of fish to polycyclic aromatic hydrocarbons (PAHs) in both field and laboratory settings has been associated with eye damage, but this has only been expressed qualitatively. In this study, an automated scanning laser system has been employed to quantitatively evaluate changes in lens optical quality in rainbow trout (Oncorhynchus mykiss) following their in vivo exposure to creosote, which is a complex mixture with many PAHs. Rainbow trout were placed in 12,000-L outdoor microcosms dosed with 0, 3, or 10 microl/L liquid creosote for a 28-day period. Collected fish were examined for changes in focal length variability (FLV), lens size, and weight. These measurements were compared with induction of hepatic ethoxyresorufin-O-deethylase (EROD) activity and hepatic and water concentrations of priority pollutant PAHs. The optical quality of rainbow trout lenses was significantly reduced following creosote exposure, as indicated by increased FLV. Lens damage was significantly related to hepatic EROD activity, and both effects rose with creosote dose. Analytical measurements of microcosm water indicated elevated concentrations of PAHs in creosote-dosed ponds, including compounds capable of inducing rainbow trout EROD activity in vitro. Hepatic concentrations of PAHs were low and not related to creosote dose, likely due to rapid metabolism and elimination. This study demonstrates for the first time employment of a highly sensitive and quantitative technique to measure lens damage in fish exposed to contaminants in situ. The relationship between this effect and hepatic CYP1A activity may suggest a mechanistic linkage, which could lead to the use of EROD activity as an indicator of toxic effect rather than just chemical exposure.

Ability of polycyclic aromatic hydrocarbons to induce 7-ethoxyresorufin-o-deethylase activity in a trout liver cell line.

Along with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 24 unsubstituted polycyclic aromatic hydrocarbons (PAHs) were evaluated for their ability to induce 7-ethoxyresorufin-o-deethylase (EROD) activity in the rainbow trout liver cell line RTL-W1. When the duration and cell density of exposure were increased, the EC(50) for EROD induction was relatively constant for TCDD, but increased for PAHs. Regardless of exposure conditions, EROD activity was not induced by 9 PAHs: naphthalene, phenanthrene, anthracene, pyrene, perylene, acenaphthylene, acenaphthene, fluorene, and fluoranthene. Two PAHs, benzo[g,h,i]perylene and coronene, induced EROD activity inconsistently. The remaining 13 PAHs consistently induced EROD activity. The EC(50)s for induction exhibited approximately a 110-fold range. The order of potency, from most to least potent, was benzo[k]fluoranthene, dibenzo[a,i]pyrene, dibenzo [a,h]anthracene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, benzo [b]fluoranthene, pentacene, benzo[b]anthracene, benzo[b] fluorene, chrysene, benzo[a]anthracene, benzo[e]pyrene, and triphenylene. When the induction potency was expressed relative to TCDD, the toxic equivalency factors (TEFs) ranged from 0.001 to 0.000 01. When expressed relative to benzo[a]pyrene, the TEFs ranged from 3.44 to 0. 03.

Treatment of hyperlipidemia by specialists versus generalists as secondary prevention of coronary artery disease.

Patients with known coronary disease and low-density lipoprotein >130 mg/dl who are followed by cardiologists after myocardial infarction are more than twice as likely to receive a cholesterol-lowering agent than patients followed by general internists.