James D. Murray

Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future

Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals’ genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611–615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

Consumption of transgenic milk containing the antimicrobials lactoferrin and lysozyme separately and in conjunction by 6-week-old pigs improves intestinal and systemic health.

Lactoferrin and lysozyme are antimicrobial and immunomodulatory proteins produced in high quantities in human milk that aid in gastrointestinal (GI) health and have beneficial effects when supplemented separately and in conjunction in human and animal diets. Ruminants produce low levels of lactoferrin and lysozyme; however, there are genetically engineered cattle and goats that respectively secrete recombinant human lactoferrin (rhLF-milk), and human lysozyme (hLZ-milk) in their milk. Effects of consumption of rhLF-milk, hLZ-milk and a combination of rhLF-and hLZ-milk were tested on young pigs as an animal model for the GI tract of children. Compared with control milk-fed pigs, pigs fed a combination of rhLF and hLZ (rhLF+hLZ) milk had a significantly deeper intestinal crypts and a thinner lamina propria layer. Pigs fed hLZ-milk, rhLF-milk and rhLF+hLZ had significantly reduced mean corpuscular volume (MCV) and red blood cells (RBCs) were significantly increased in pigs fed hLZ-milk and rhLF-milk and tended to be increased in rhLF+hLZ-fed pigs, indicating more mature RBCs. These results support previous research demonstrating that pigs fed milk containing rhLF or hLZ had decreased intestinal inflammation, and suggest that in some parameters the combination of lactoferrin and lysozyme have additive effects, in contrast to the synergistic effects reported when utilising in-vitro models.

The lactoferrin receptor may mediate the reduction of eosinophils in the duodenum of pigs consuming milk containing recombinant human lactoferrin

Lactoferrin is part of the immune system and multiple tissues including the gastrointestinal (GI) tract, liver, and lung contain receptors for lactoferrin. Lactoferrin has many functions, including antimicrobial, immunomodulatory, and iron binding. Additionally, lactoferrin inhibits the migration of eosinophils, which are constitutively present in the GI tract, and increase during inflammation. Lactoferrin suppresses eosinophil infiltration into the lungs and eosinophil migration in -vitro. Healthy pigs have a large population of eosinophils in their small intestine and like humans, pigs have small intestinal lactoferrin receptors (LFR); thus, pigs were chosen to investigate the effects of consumption of milk containing recombinant human lactoferrin (rhLF-milk) on small intestinal eosinophils and expression of eosinophilic cytokines. In addition, LFR localization was analyzed in duodenum and circulating eosinophils to determine if the LFR could play a role in lactoferrin’s ability to inhibit eosinophil migration. In the duodenum there were significantly fewer eosinophils/unit area in pigs fed rhLF-milk compared to pigs fed control milk (pxa0=xa00.025); this was not seen in the ileum (pxa0=xa00.669). In the duodenum, no differences were observed in expression of the LFR, or any eosinophil migratory cytokines, and the amount of LFR protein was not different (pxa0=xa00.386). Immunohistochemistry (IHC) showed that within the duodenum the LFR localized on the brush border of villi, crypts, and within the lamina propria. Circulating eosinophils also contained LFRs, which may be a mechanism allowing lactoferrin to directly inhibit eosinophil migration.

Genetically Engineered Livestock: Ethical Use for Food and Medical Models

Recent advances in the production of genetically engineered (GE) livestock have resulted in a variety of new transgenic animals with desirable production and composition changes. GE animals have been generated to improve growth efficiency, food composition, and disease resistance in domesticated livestock species. GE animals are also used to produce pharmaceuticals and as medical models for human diseases. The potential use of these food animals for human consumption has prompted an intense debate about food safety and animal welfare concerns with the GE approach. Additionally, public perception and ethical concerns about their use have caused delays in establishing a clear and efficient regulatory approval process. Ethically, there are far-reaching implications of not using genetically engineered livestock, at a detriment to both producers and consumers, as use of this technology can improve both human and animal health and welfare.

Tissue resolved, gene structure refined equine transcriptome

BackgroundTranscriptome interpretation relies on a good-quality reference transcriptome for accurate quantification of gene expression as well as functional analysis of genetic variants. The current annotation of the horse genome lacks the specificity and sensitivity necessary to assess gene expression especially at the isoform level, and suffers from insufficient annotation of untranslated regions (UTR) usage. We built an annotation pipeline for horse and used it to integrate 1.9 billion reads from multiple RNA-seq data sets into a new refined transcriptome.ResultsThis equine transcriptome integrates eight different tissues from 59 individuals and improves gene structurexa0and isoform resolution, while providing considerable tissue-specific information. We utilized four levels of transcript filtration in our pipeline, aimed at producing several transcriptome versions that are suitable for different downstream analyses. Our most refined transcriptome includes 36,876 genes and 76,125 isoforms, with 6474 candidate transcriptional loci novel to the equine transcriptome.ConclusionsWe have employed a variety of descriptive statistics and figures that demonstrate the quality and content of the transcriptome. The equine transcriptomes that are provided by this pipeline show the best tissue-specific resolution of any equine transcriptome to date and are flexible for several downstream analyses. We encourage the integration of further equine transcriptomes with our annotation pipeline to continue and improve the equine transcriptome.

Genetically engineered livestock for agriculture: a generation after the first transgenic animal research conference

At the time of the first Transgenic Animal Research Conference, the lack of knowledge about promoter, enhancer and coding regions of genes of interest greatly hampered our efforts to create transgenes that would express appropriately in livestock. Additionally, we were limited to gene insertion by pronuclear microinjection. As predicted then, widespread genome sequencing efforts and technological advancements have profoundly altered what we can do. There have been many developments in technology to create transgenic animals since we first met at Granlibakken in 1997, including the advent of somatic cell nuclear transfer-based cloning and gene editing. We can now create new transgenes that will express when and where we want and can target precisely in the genome where we want to make a change or insert a transgene. With the large number of sequenced genomes, we have unprecedented access to sequence information including, control regions, coding regions, and known allelic variants. These technological developments have ushered in new and renewed enthusiasm for the production of transgenic animals among scientists and animal agriculturalists around the world, both for the production of more relevant biomedical research models as well as for agricultural applications. However, even though great advancements have been made in our ability to control gene expression and target genetic changes in our animals, there still are no genetically engineered animal products on the market for food. World-wide there has been a failure of the regulatory processes to effectively move forward. Estimates suggest the world will need to increase our current food production 70xa0% by 2050; that is we will have to produce the total amount of food each year that has been consumed by mankind over the past 500xa0years. The combination of transgenic animal technology and gene editing will become increasingly more important tools to help feed the world. However, to date the practical benefits of these technologies have not yet reached consumers in any country and in the absence of predictable, science-based regulatory programs it is unlikely that the benefits will be realized in the short to medium term.

Milk with and without lactoferrin can influence intestinal damage in a pig model of malnutrition

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of five worldwide. However, the underlying mechanisms are not well understood necessitating an appropriate animal model to answer fundamental questions and conduct translational research into optimal interventions. One potential intervention is milk from livestock that more closely mimics human milk by increased levels of bioactive components that can promote a healthy intestinal epithelium. We tested the ability of cow milk and milk from transgenic cows expressing human lactoferrin at levels found in human milk (hLF milk) to mitigate the effects of malnutrition at the level of the intestine in a pig model of malnutrition. Weaned pigs (3 weeks old) were fed a protein and calorie restricted diet for five weeks, receiving cow, hLF or no milk supplementation daily from weeks 3-5. After three weeks, the restricted diet induced changes in growth, blood chemistry and intestinal structure including villous atrophy, increased ex vivo permeability and decreased expression of tight junction proteins. Addition of both cow and hLF milk to the diet increased growth rate and calcium and glucose levels while promoting growth of the intestinal epithelium. In the jejunum hLF milk restored intestinal morphology, reduced permeability and increased expression of anti-inflammatory IL-10. Overall, this pig model of malnutrition mimics salient aspects of the human condition and demonstrates that cow milk can stimulate the repair of damage to the intestinal epithelium caused by protein and calorie restriction with hLF milk improving this recovery to a greater extent.

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.

Identification of long non-coding RNA in the horse transcriptome

BackgroundEfforts to resolve the transcribed sequences in the equine genome have focused on protein-coding RNA. The transcription of the intergenic regions, although detected via total RNA sequencing (RNA-seq), has yet to be characterized in the horse. The most recent equine transcriptome based on RNA-seq from several tissues was a prime opportunity to obtain a concurrent long non-coding RNA (lncRNA) database.ResultsThis lncRNA database has a breadth of eight tissues and a depth of over 20 million reads for select tissues, providing the deepest and most expansive equine lncRNA database. Utilizing the intergenic reads and three categories of novel genes from a previously published equine transcriptome pipeline, wexa0better describe these groups by annotating the lncRNA candidates. These lncRNA candidates were filtered using an approach adapted from human lncRNA annotation, which removes transcripts based on size, expression, protein-coding capability and distance to the start or stop of annotated protein-coding transcripts.ConclusionOur equine lncRNA database has 20,800 transcripts that demonstrate characteristics unique to lncRNA including low expression, low exon diversity and low levels of sequence conservation. These candidate lncRNA will serve as a baseline lncRNA annotation and begin to describe the RNA-seq reads assigned to the intergenic space in the horse.

Transgenic animal models for the production of human immunocompounds in milk to prevent diarrhea, malnourishment and child mortality: perspectives for the Brazilian Semi-Arid region

Diarrhea is one of the most significant issues for global health in children under the age of five. Close to 60% of mortality in this risk group is caused by pneumonia, diarrhea or measles, which is usually associated with malnutrition. In Brazil, this scenario is no different, with the Northeast semi-arid region having alarming rates of child mortality, almost twice that of the current national mortality rate, with some cities being three times higher, placing child mortality rates in the Northeast of Brazil among those of the most problematic in the world. The oral rehydration solution, introduced by the World Health Organization (WHO), is seen as one of the major medical advances in the past 50 years, and is believed to save the lives of 1 to 2 million children each year. However, the oral rehydration solution simply restores body fluid normality, treating the consequences and not the causes of diarrheas. Other means to effectively address worldwide diarrheic ailments still need to be devised or improved, focusing more on preventive rather than curative approaches, to prevent or shorten the course of disease and to minimize recurrences. A great example is breast-feeding. A series of studies documented the reduction of diarrhea episodes in breast-fed children and, also, noted a faster recovery time following an infection in these children. The positive benefit of breast milk is attributed to the antimicrobial actions of human milk proteins, such as lysozyme and lactoferrin that can enhance intestinal and systemic immunological functions. Unfortunately, breast-feeding is not always an option, especially for toddlers, which is usually aggravated by undernourishment and low standard of living, reinforcing the need for alternative strategies. Consequently, to successfully combat childhood undernourishment, morbidity and mortality in the Northeast region of Brazil, the investment in approaches and models that are more suitable to the local semi-arid adapted agriculture-base is required. The goat is an important economic asset to the Northeast region, providing milk, meat and leather, with the number of goats in the Northeast region representing more than 90% of the Brazilian herd. Historically, goats have been used as a model in biotechnological studies involving the expression of specific proteins in the milk of lactating animals. In this way, the use of goats expressing recombinant human lysozyme (rhLZ) and lactoferrin (rhLF) in milk is a proven reality that can be employed for the reduction of malnutrition, infectious diseases and diarrhea in children in the Brazilian semi-arid region with an extraordinary potential for success. Due to the protective nature of human milk imparted, in part, by the presence of lysozyme and lactoferrin, the generation of transgenic lines for the production of these important antimicrobial compounds in the milk of the local goat population is a unique approach for delivering the potential protective benefits of human milk. In addition, the use of rhLZ and rhLF transgenic animals can be used as a research tool for studying and elucidating the individual roles of these antimicrobial proteins in gut development and their synergistic in vivo effects. The proposed research was designed with the rational that the transgenic approach can be used to modify the milk composition of dairy animals to supply milk-borne human immunocompounds (rhLZ, rhLF) and nutrients University of Fortaleza, UNIFOR, Fortaleza, Brazil Full list of author information is available at the end of the article Bertolini et al. BMC Proceedings 2014, 8(Suppl 4):O30 http://www.biomedcentral.com/1753-6561/8/S4/O30