DNA methylation as a regulator of intestinal gene expression.

The intestinal tract is the entry gate for nutrients and symbiotic organisms, being in constant contact with external environment. DNA methylation is one of the keys to how environmental conditions, diet and nutritional status included, shape functionality in the gut and systemically. This review aims to summarise findings on the importance of methylation to gut development, differentiation and function. Evidence to date on how external factors such as diet, dietary supplements, nutritional status and microbiota modifications modulate intestinal function through DNA methylation is also presented.

Systemic immunosuppression by methylprednisolone and pregnancy rates in goats undergoing the transfer of cloned embryos.

The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona-free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre-treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona-free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo- vs in vitro-matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona-free and zona-intact goat cloned embryos.

Short communication: Identification of coagulase-negative staphylococcus species from goat milk with the API Staph identification test and with transfer RNA-intergenic spacer PCR combined with capillary electrophoresis.

Coagulase-negative staphylococci (CNS) are the most commonly isolated bacteria from goat milk, but they have often been identified with phenotypic methods, which may have resulted in misclassification. The aims of this paper were to assess the amount of misclassification of a phenotypic test for identifying CNS species from goat milk compared with transfer RNA intergenic spacer PCR (tDNA-PCR) followed by capillary electrophoresis, and to apply the tDNA-PCR technique on different capillary electrophoresis equipment. Milk samples were collected from 416 does in 5 Californian dairy goat herds on 3 occasions during lactation. In total, 219 CNS isolates were identified at the species level with tDNA-PCR and subjected to the API 20 Staph identification test kit (API Staph; bioMérieux, Durham, NC). If the same species was isolated multiple times from the same udder gland, only the first isolate was used for further analyses, resulting in 115 unique CNS isolates. According to the tDNA-PCR test, the most prevalent CNS species were Staphylococcus epidermidis, Staphylococcus caprae, and Staphylococcus simulans. Typeability with API staph was low (72%). Although the API Staph test was capable of identifying the majority of Staph. epidermidis and Staph. caprae isolates, sensitivity for identification of Staph. simulans was low. The true positive fraction was high for the 3 most prevalent species. It was concluded that the overall performance of API Staph in differentiating CNS species from goat milk was moderate to low, mainly because of the low typeability, and that genotypic methods such as tDNA-PCR are preferred.

Precision genetics for complex objectives in animal agriculture.

Indirect modification of animal genomes by interspecific hybridization, cross-breeding, and selection has produced an enormous spectrum of phenotypic diversity over more than 10,000 yr of animal domestication. Using these established technologies, the farming community has successfully increased the yield and efficiency of production in most agricultural species while utilizing land resources that are often unsuitable for other agricultural purposes. Moving forward, animal well-being and agricultural sustainability are moral and economic priorities of consumers and producers alike. Therefore, these considerations will be included in any strategy designed to meet the challenges produced by global climate change and an expanding world population. Improvements in the efficiency and precision of genetic technologies will enable a timely response to meet the multifaceted food requirements of a rapidly increasing world population.

Welfare applications of genetically engineered animals for use in agriculture.

The application of genetic engineering to food animals is often viewed as a means to further increase animal productivity without regard for the welfare of the resulting animals. We offer the perspective that, on the contrary, genetic engineering can, and is, being used to improve animal welfare in modern production systems. Several examples are cited from the current work in the field of animal genetic engineering that should be included in the debate over whether genetically engineered animals should be used in production agriculture. The current debate has slowed the advancement of this technology, which could play a key role in improving animal welfare and sustainability, without considering the potential benefits.

Prevalence of alphas1-casein genotypes in American dairy goats.

Widespread genotyping of US dairy goat breeds for casein variants has not been reported, even though the genetic data could be of use in selective breeding programs. For instance, variability in the content of protein and solids in goat milk is attributed to allelic differences in the goat alpha(s1)-casein gene. Concentrations of alpha(s1)-casein in goat milk are positively correlated with milk components and coagulation properties. The alleles A and B are designated as strong alleles, resulting in the greatest amount of alpha(s1)-casein in goat milk, whereas the E allele produces intermediate amounts and the weak allele F produces the least concentrations of alpha(s1)-casein in goat milk. Here we report on one of the first surveys of the distribution of alpha(s1)-casein genotypes in US dairy goats. The population surveyed, consisting of a total of 257 American dairy goats representing 7 main dairy breeds, contained a greater predominance of the weaker alleles, E and F, than the strong alleles, A and B. Allele distribution was related to breed, with Toggenburg, Alpine, Saanen, and Oberhasli containing the most E and F alleles and LaMancha, Nubian, and Nigerian Dwarf the fewest. Quantification of alpha(s1)-casein production by 2-dimensional gel electrophoresis demonstrated that F/F animals had the least amount of alpha(s1)-casein protein in their milk compared with all other genotypes. The results indicate that genetic improvement of dairy goats in the United States could be achieved if an alpha(s1)-casein breeding scheme were adopted.

Use of human lysozyme transgenic goat milk in cheese making: effects on lactic acid bacteria performance.

Genetically engineered goats expressing elevated levels of the antimicrobial enzyme lysozyme in their milk were developed to improve udder health, product shelf life, and consumer well-being. The purpose of this study was to evaluate the effect of lysozyme on the development of lactic acid bacteria (LAB) throughout the cheese-making process. Raw and pasteurized milk from 7 lysozyme transgenic goats and 7 breed-, age-, and parity-matched nontransgenic controls was transformed into cheeses by using industry methods, and their microbiological load was evaluated. The numbers of colony-forming units of LAB were determined for raw and pasteurized goat milk, whey, and curd at d 2 and at d 6 or 7 of production. Selective plating media were used to enumerate lactococcal species separately from total LAB. Although differences in the mean number of colony-forming units between transgenic and control samples in raw milk, whey, and cheese curd were non-significant for both total LAB and lactococcal species from d 2 of production, a significant decrease was observed in both types of LAB among d 6 transgenic raw milk cheese samples. In pasteurized milk trials, a significant decrease in LAB was observed only in the raw milk of transgenic animals. These results indicate that lysozyme transgenic goat milk is not detrimental to LAB growth during the cheese-making process.

Production and processing of milk from transgenic goats expressing human lysozyme in the mammary gland.

The potential for applying biotechnology to benefit animal agriculture and food production has long been speculated. The addition of human milk components with intrinsic antimicrobial activity and positive charge to livestock milk by genetic engineering has the potential to benefit animal health, as well as food safety and production. We generated one line of transgenic goats as a model for the dairy cow designed to express human lysozyme in the mammary gland. Here we report the characterization of the milk from 5 transgenic females of this line expressing human lysozyme in their milk at 270 microg/mL or 68% of the level found in human milk. Milk from transgenic animals had a lower somatic cell count, but the overall component composition of the milk and milk production were not different from controls. Milk from transgenic animals had a shorter rennet clotting time and increased curd strength. Milk of such nature may be of benefit to the producer by influencing udder health and milk processing.

Hot topic: using a stearoyl-CoA desaturase transgene to alter milk fatty acid composition.

Stearoyl-CoA desaturase enzyme converts specific medium- and long-chain saturated fatty acids to their monounsaturated form. Transgenic goats expressing a bovine beta-lactoglobulin promoter-rat stearoyl-CoA desaturase cDNA construct in mammary gland epithelial cells were produced by pronuclear microinjection. The fatty acid composition of milk from 4 female transgenic founders was analyzed on d 7, 14, and 30 of their first lactation. In 2 animals, the expression of the transgene changed the overall fatty acid composition of the resulting milk fat to a less saturated and more monounsaturated fatty acid profile at d 7 of lactation; however, this effect diminished by d 30. In addition, one animal had an increased proportion of the rumen-derived monounsaturated fatty acid C18:1 trans11 converted by stearoyl-CoA desaturase to the conjugated linoleic acid isomer C18:2 cis9 trans11. Milk that has higher proportions of monounsaturated fatty acids and conjugated linoleic acid may have benefits for human cardiovascular health.

The use of recombinase proteins to generate transgenic large animals.

The endogenous properties of recombinase proteins allow them to associate with and bind DNA to catalyze homologous recombination. These endogenous properties of cellular recombination enzymes may be useful to the field of transgenesis. The production of transgenic animals, in particular livestock, is an inefficient process by both conventional pronuclear microinjection techniques and nuclear transfer. Furthermore, the use of pronuclear microinjection is currently limited to the random addition of genes and does not allow for the replacement of an endogenous gene with a more desired one. The functions of cellular recombination enzymes have been exploited to develop a technique that is compatible with pronuclear microinjection and may make the process of generating transgenic livestock more efficient while also enabling the targeting of homologous chromosomal genes. In our hands, transgenic animals generated by the pronuclear microinjection of various recombinase protein-coated DNA fragments led to a higher than expected birth rate as well as transgene integration frequency. Most founder animals generated were likely mosaic, indicating that integration occurred after cell division. The presence of multiple related genes makes detection of any recombination event difficult. Overall, this technique is a straightforward, rapid, and efficient procedure that can be applied to any segment of DNA and any microinjection apparatus, and is less labor intensive than nuclear transfer.

Antimicrobial properties of human lysozyme transgenic mouse milk.

The antimicrobial properties of standard human lysozyme and the milk of transgenic mice expressing human lysozyme were investigated using bacterial strains important to the dairy industry. Standard human lysozyme was found to be effective at significantly slowing the growth of the milk cold-spoilage organism Pseudomonas fragi (P < 0.001), of a clinical isolate of the mastitis-causing organism Staphylococcus aureus (P < 0.005), and a nonpathogenic strain of E. coli (P < 0.05). Milk from transgenic mice secreting human lysozyme in their milk at an average concentration of 0.3 mg/ml was found to be bacteriostatic against the cold-spoilage organisms Pseudomonas fragi and Lactobacillus viscous and a mastitis-causing strain of Staphylococcus aureus, but not against a pathogenic strain of E. coli. These results demonstrate that transgenic animals producing human lysozyme in their milk can affect the microbial nature of milk.

MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells.

Proteins comprising the mitogen-activated protein (MAP) kinase signaling cascade are activated by a variety of growth factors, but the precise role of this series of kinase reactions, especially Raf kinase and MAP kinase kinase (MEK), in vascular smooth muscle (VSM) cell mitogenesis is not known. In this study, a specific and selective inhibitor of MEK, PD-98059, was used to examine the role of MEK in DNA synthesis and Raf-1 activity in VSM cells stimulated with serum as well as with growth factors encompassing both tyrosine kinase and G protein-coupled receptor classes. Although significant increases in DNA synthesis are seen after stimulation of VSM cells with either 10% serum,platelet-derived growth factor (PDGF)-BB, or alpha-thrombin, preincubation of the cells with 50 microM PD-98059 for 1 h inhibits stimulation by PDGF and thrombin, but not by serum. There is a dose-dependent inhibition of the mitogenic effect by PD-98059 in all cases; these results are not affected when PD-98059 is added at times ranging from 4 h before to 2 h after growth factor addition (times at which PD-98059 exerts its inhibitory effect). In the presence of PD-98059, stimulated MAP kinase activity is attenuated when growth factors are added between 10 min and 4 h, times which correspond to both early and sustained phases of MAP kinase activity. In addition, Raf-1 activity is markedly increased by incubation of the cells with PD-98059,despite attenuation of hyperphosphorylation of this kinase. Thus growth factors coupled to both tyrosine kinase and G protein receptors require components of the MAP kinase cascade (MEK and/or MAP kinase) for VSM cell mitogenesis, whereas serum is capable of stimulatory effects in the absence of active MEK and MAP kinase. Furthermore, there exists a functional feedback stimulatory effect of inhibited MEK on its upstream activator Raf-1 in the case of serum as well as growth factors coupled to tyrosine kinase and G protein receptors.

Alterations of the physical characteristics of milk from transgenic mice producing bovine kappa-casein.

kappa-Casein is the protein fraction of milk that allows formation of micelles and determines micelle size and function, thus affecting many of the physical characteristics of milk. Several lines of transgenic mice were generated bearing the B allele of the bovine kappa-CN gene under the control of the regulatory sequences of the caprine beta-CN gene that specifically directed expression of bovine kappa-CN to the lactating mammary tissue of these mice. High expression of bovine kappa-CN protein was observed in the lines studied; the total level of protein in milk was not significantly affected. A high degree of conservation in the amino acids involved in the predicted three-dimensional structure exists between murine and bovine kappa-CN. Milk from transgenic lines expressing high bovine kappa-CN had a significantly smaller micelle size than did control milk. Therefore, bovine kappa-CN appears to have effectively participated in assembly of murine casein micelles. There was no effect on the time of rennet coagulation, but the association was significant between the milk of transgenic lines and the production of a stronger curd in rennet-induced gels. We conclude that bovine kappa-CN is an appropriate candidate for transgenic technology that would increase the ratio of kappa-CN to the calcium-sensitive caseins, therefore affecting the physical properties of the colloidal casein suspension.

The effect of mammary gland expression of human lysozyme on the properties of milk from transgenic mice.

Transgenic mice were used as model systems to evaluate the impact of human lysozyme expression in the mammary gland. We previously generated two lines of transgenic mice that express human lysozyme mRNA in the mammary gland under the tissue-specific and developmentally correct control of the bovine gene promoter for alpha s1-casein. Concentrations of human lysozyme protein in milk of transgenic mice varied from .25 to .71 micrograms/microliters of milk. Human lysozyme secreted into mouse milk retained its antimicrobial activity, as determined by a denaturing polyacrylamide gel activity assay. The physical and functional properties of the milk were also altered, because mouse milk containing human lysozyme had a 35% decrease in rennet clotting time, a smaller median micelle size (157 nm vs. 172 nm), and a 2.5- to 3-fold greater gel strength than control milk. From these results, we conclude that the use of transgenic animals producing lysozyme in the milk is feasible and potentially useful to the dairy industry.

Mammary gland expression of transgenes and the potential for altering the properties of milk.

Transgenic animals are a useful in vivo experimental model for assessing the ability and impact of foreign gene expression in a biological system. Transgenic mice are most commonly used, while transgenic sheep, goats, pigs and cows have also been developed for specific, "applied" purposes. Most of the work directed at targeting expression of transgenes to the mammary gland of an animal, by using a milk gene promoter, has been with the intent of either studying promoter function or recovering the desired protein from the milk. Transgenic technology can also be used to alter the functional and physical properties of milk resulting in novel manufacturing properties. The properties of milk have been altered by adding a new protein with the aim of improving the milk, not of recovering the protein for other uses.

Expression of human lysozyme mRNA in the mammary gland of transgenic mice.

Owing to its inherent antimicrobial effect and positive charge, the expression of human lysozyme in bovine milk could be beneficial by altering the overall microbial level and the functional and physical properties of the milk. We have used transgenic mice as model systems to evaluate the expression of human lysozyme containing fusion gene constructs in the mammary gland. Expression of human lysozyme was targeted to the mammary gland by using the 5' promoter elements of either the bovine beta (line B mice) or alpha s1 (line H mice) casein genes coupled to the cDNA for human lysozyme. Expression of human lysozyme mRNA was not found in mammary tissue from any of line B mice. Tissues were analysed from six lines of H mice and two, H6 and H5, were found to express human lysozyme mRNA in the mammary gland at 42% and 116%, respectively, of the levels of the endogenous mouse whey acidic protein gene. At peak lactation, female mice homozygous for the H5 and H6 transgene have approximately twice the amount of mRNA encoding human lysozyme as hemizygous animals. Expression levels of human lysozyme mRNA in the mammary gland at time points representing late pregnancy, early, peak and late lactation corresponded to the profile of casein gene expression. Human lysozyme mRNA expression was not observed in transgenic males, virgin females or in the kidney, liver, spleen or brain of lactating females. A very low level of expression of human lysozyme mRNA was observed in the salivary gland of line H5.