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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.

The Wnt signaling pathway is critical for normal embryonic development. Disruptions in the Wnt signaling pathway have been linked to neurological disorders. The RAPGEF5 protein is a partner in Wnt signaling and a RAPGEF5 3-bp insertion is associated with increased risk for idiopathic epilepsy in the Belgian shepherd dog. The 3-bp insertion risk variant introduces an alanine residue predicted to disrupt the protein. Wildtype and the risk variant RAPGEF5 cDNAs were cloned into green fluorescent protein (GFP) expression vectors and transfected into canine kidney cells. The cellular localization of each GFP-labeled RAPGEF5 protein was assessed. Variant RAPGEF5 protein was altered in its localization from that of the wildtype protein and rather than localized to the nucleus and cytoplasm as seen for the wildtype, it was predominantly found in the cytoplasm. Belgian shepherds with the risk variant for RAPGEF5 may have altered Wnt signaling due to modified intracellular localization which in turn could thereby contribute to the expression of idiopathic epilepsy., (© 2024. The Author(s).)

Background: The mare-foal relationship is essential for the well-being and growth of a foal. Mare's milk provides a foal with nutrients, protective immunity, and microbes. Within the first two weeks of life, there is a risk for a foal to suffer from diarrhea, particularly "foal heat diarrhea" which happens at about the time of a mare's estrus cycle but is more likely due to transitions in the microbiota in the foal's gastrointestinal (GI) tract. We hypothesized that this GI microbiota transition could be caused by changes in lysozyme and microbial populations in the mare's milk. To test this hypothesis, fifteen mare-foal pairs were followed in the first 15 days post-foaling. Every other day milk was collected from mares and rectal swabs were collected from foals. Lysozyme activity in the mare's milk was measured using a fluorescence assay. Microbial DNA was isolated from the milk and swabs and the V4 domain of 16 S rRNA genes were PCR amplified and sequenced using Illumina MiSeq technology. Microbial populations were analyzed using DADA2 and phyloseq within R., Results: Mare's milk lysozyme activity peaked for samples at Day 1 and levels dropped to 72.5% of Day 1 activity by Day 15; however, microbial populations in the mare's milk did not vary significantly over the two weeks. Furthermore, levels of microbial diversity found in foal rectal swabs were initially similar to microbial diversity seen in mare's milk; however, over the first fifteen days, diversity increased for the foal rectal swab microbiota and swab microbial populations differed from milk microbes. A transition occurred shifting from microbes from the phylum Proteobacteria early in rectal swabs to those primarily from the phyla Firmicutes and Bacteroidota after the first few days post-foaling. These phyla contained several families and genera of microbes that promote utilization of milk components in healthy gut transition. Microbial abundance levels correlated more with days post-parturition than with lysozyme activity and mare's milk microbial populations., Conclusions: The findings suggest that much of the microbial populations responsible for the transition of the foal's gut comes from sources outside of mare's milk species and levels of lysozyme activity., (© 2024. The Author(s).)

A single locus on the X chromosome codes for androgen receptor (AR) although this gene is subject to alternative splicing. AR is expressed in multiple tissues in males and females and is essential for reproductive success in the male. Since male and female mice are viable following naturally occurring and engineered loss of function with male mice infertile as anticipated, functional deletion of AR in pigs was hypothesized to provide a genetic containment strategy for males with edited genomes. In addition, deletion of AR might be a method to manage boar taint, hence contributing to a perceived improvement in animal welfare. The CRISPR/Cas9 technology was used to edit either exon 2 or exon 5 of the pig AR gene. Although pregnancies were established following embryo transfer of edited embryos, they were not maintained beyond day 25. Furthermore, normal M:F sex ratios were present in edited blastocysts and 19-day fetuses, but all fetuses recovered on day 21 or later were female. The pig AR gene differs from the mouse in having a U2 spliceosome component encoded in the intronic region. Hence, the absence of fetal survival beyond day 25 may be due to interference with the U2 component rather than AR., (© 2023. Springer Nature Limited.)

Tendinopathies remain the leading contributor to career-ending injuries in horses because of the complexity of tendon repair. As such, cell-based therapies like injections of adipose-derived mesenchymal stem cells (ADMSCs, or MSCs) into injured tendons are becoming increasingly popular though their long-term efficacy on a molecular and wholistic level remains contentious. Thus, we co-cultured equine MSCs with intrinsic (tendon proper) and extrinsic (peritenon) tendon cell populations to examine interactions between these cells. Gene expression for common tenogenic, perivascular, and differentiation markers was quantified at 48 and 120 hours. Additionally, cellular metabolism of proliferation was examined every 24 hours for peritenon and tendon proper cells co-cultured with MSCs. MSCs co-cultured with tendon proper or peritenon cells had altered expression profiles demonstrating trend toward tenogenic phenotype with the exception of decreases in type I collagen (COL1A1). Peritenon cells co-cultured with MSCs had a trending and significant decrease in biglycan (BGN) and CSPG4 at 48 hours and 120 hours but overall significant increases in lysyl oxidase (LOX), mohawk (MKX), and scleraxis (SCX) within 48 hours. Tendon proper cells co-cultured with MSCs also exhibited increases in LOX and SCX at 48 hours. Furthermore, cell proliferation improved overall for tendon proper cells co-cultured with MSCs. The co-culture study results suggest that adipose-derived MSCs contribute beneficially to tenogenic stimulation of peritenon or tendon proper cells., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Malnourishment is a risk factor for childhood mortality, jeopardizing the health of children by aggravating pneumonia/acute respiratory infections and diarrheal diseases. Malnourishment causes morphophysiological changes resulting in stunting and wasting that have long-lasting consequences such as cognitive deficit and metabolic dysfunction. Using a pig model of malnutrition, the interplay between the phenotypic data displayed by the malnourished animals, the gene expression pattern along the intestinal tract, microbiota composition of the intestinal contents, and hepatic metabolite concentrations from the same animals were correlated using a multi-omics approach. Samples from the duodenum, jejunum, and ileum of malnourished (protein and calorie-restricted diet) and full-fed (no dietary restrictions) piglets were subjected to RNA-seq. Gene co-expression analysis and phenotypic correlations were made with WGCNA, while the integration of transcriptome with microbiota composition and the hepatic metabolite profile was done using mixOmics. Malnourishment caused changes in tissue gene expression that influenced energetic balance, cell proliferation, nutrient absorption, and response to stress. Repression of antioxidant genes, including glutathione peroxidase, in coordination with induction of metal ion transporters corresponded to the hepatic metabolite changes. These data indicate oxidative stress in the intestine of malnourished animals. Furthermore, several of the phenotypes displayed by these animals could be explained by changes in gene expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pinho, Garas, Huang, Weimer and Maga.)

The rumen is a complex ecosystem that plays a critical role in our efforts to improve feed efficiency of cattle and reduce their environmental impacts. Sequencing of the 16S rRNA gene provides a powerful tool to survey the bacterial and some archaeal. Oral stomach tubing a cow to collect a rumen sample is a rapid, cost-effective alternative to rumen cannulation for acquiring rumen samples. In this study, we determined how sampling method (oral stomach tubing vs cannulated grab sample), as well as rumen fraction type (liquid vs solid), bias the bacterial and archaeal communities observed. Liquid samples were further divided into liquid strained through cheesecloth and unstrained. Fecal samples were also collected to determine how these differed from the rumen sample types. The abundance of major archaeal communities was not different at the family level in samples acquired via rumen cannula or stomach tube. In contrast to the stable archaeal communities across sample type, the bacterial order WCHB1-41 (phylum Kiritimatiellaeota) was enriched in both liquid strained and unstrained samples as well as the family Prevotellaceae as compared to grab samples. However, these liquid samples had significantly lower abundance of Lachnospiraceae compared with grab samples. Solid samples strained of rumen liquid most closely resembled the grab samples containing both rumen liquid and solid particles obtained directly from the rumen cannula; therefore, inclusion of particulate matter is important for an accurate representation of the rumen bacteria. Stomach tube samples were the most variable and were most representative of the liquid phase. In comparison with a grab sample, stomach tube samples had significantly lower abundance of Lachnospiraceae, Fibrobacter and Treponema. Fecal samples did not reflect the community composition of the rumen, as fecal samples had significantly higher relative abundance of Ruminococcaceae and significantly lower relative abundance of Lachnospiraceae compared with grab samples., Competing Interests: The authors have declared that no competing interests exist.

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.

Severe acute malnutrition (SAM), due to poor energy and/or protein intake, is associated with poor growth, depressed immune function, and long-term impacts on metabolic function. As the liver is a major metabolic organ and malnutrition poses metabolic stress, we hypothesize that SAM will be associated with alterations in the hepatic metabolome reflective of oxidative stress, gluconeogenesis, and ketogenesis. Thus, the purpose of this secondary analysis was to understand how SAM alters hepatic metabolism using a piglet model. Weanling piglets were feed either a reference (REF) or protein-energy deficient diet (MAL) for 5 weeks. After dietary treatment MAL piglets were severely underweight (weight-for-age Z-score of -3.29, Welch's t test, P = .0007), moderately wasted (weight-for-length Z-score of-2.49, Welch's t test, P = .003), and tended toward higher hepatic triglyceride content (Welch's t test, P = .07). Hematologic and blood biochemical measurements were assessed at baseline and after dietary treatment. The hepatic metabolome was investigated using 1 H-NMR spectroscopy. Hepatic concentrations of betaine, cysteine, and glutathione tended to be lower in MAL (Welch's t test with FDR correction, P < .1), while inosine, lactate, and methionine sulfoxide concentrations were higher in MAL (inosine: P = .0448, lactate: P = .0258, methionine sulfoxide: P = .0337). These changes suggest that SAM is associated with elevated hepatic oxidative stress, increased gluconeogenesis, and alterations in 1-carbon metabolism., (Copyright © 2021. Published by Elsevier Inc.)

Objective: Prophylactic supplementation of psyllium husk is recommended to enhance passage of ingested sand from the gastrointestinal tracts of horses. We hypothesized that psyllium supplementation would increase fecal sand passage and favorably alter bacterial populations in the hindgut. Six yearlings and six mature mares were fed a psyllium supplement in the diet daily for seven days. Voluntarily-voided feces were collected over the course of 29 days, prior, during, and after treatment. Feces were analyzed for acid detergent fiber (ADF) and acid detergent insoluble ash analyses. Microbial DNA was also isolated, and the V4 region of the 16S ribosomal RNA gene was PCR-amplified and sequenced using MiSeq technology., Results: Fecal ADF concentration was greater in adults while silica concentration was greater in yearlings. Mature mare fecal ADF decreased during and just after supplementation but thereafter increased. No changes in silica levels were noted in either group over time. Fecal microbial population phylogenetic diversity was greatest mid-supplementation and lowest at 11 days post-supplementation. Functional profiles of the microbial communities presented some benefits for psyllium supplementation. These findings provide compelling evidence for further detailed studies of prophylactic psyllium supplementation.

This experiment aimed to evaluate meat quality, fatty acid profile in back-fat, and fecal microbiota of growing-finishing pigs fed with liquid enzymatically digested food waste. Fifty-six crossbred pigs (approximately 32.99 kg body weight) were assigned to one of two treatments with seven replicate pens and four pigs per pen. Pigs were fed with control (corn-soybean meal diets) or food waste from d 0 to 53, while all pigs were fed with the control diet from d 53 to 79. The 16S rRNA sequencing was used to analyze microbiota of feces collected on d 0, 28, 53, and 79. Meat quality and carcass characteristics were measured in one pig per pen at the end of the experiment. Pigs fed with food waste contained more ( p < 0.05) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in back-fat. Feeding food waste increased ( p < 0.05) the relative abundances of Lachnospiraceae and Ruminococcaceae , but decreased ( p < 0.05) the relative abundances of Streptococcaceae and Clostridiaceae in feces on d 29 or d 53. In conclusion, feeding enzymatically digested food waste did not affect pork quality, but provided more beneficial fatty acids to pork consumers and altered the fecal microbiota in growing-finishing pigs.

Improved sequencing and analytical techniques allow for better resolution of microbial communities; however, the agriculture field lacks an updated analysis surveying the fecal microbial populations of dairy cattle in California. This study is a large-scale survey to determine the composition of the bacterial community present in the feces of lactating dairy cattle on commercial dairy operations. For the study, 10 dairy farms across northern and central California representing a variety of feeding and management systems were enrolled. The farms represented three typical housing types including five freestall, two drylot and three pasture-based management systems. Fresh feces were collected from 15 randomly selected cows on each farm and analyzed using 16S rRNA gene amplicon sequencing. This study found that housing type, individual farm, and dietary components significantly affected the alpha diversity of the fecal microbiota. While only one Operational Taxonomic Unit (OTU) was common among all the sampled individuals, 15 bacterial families and 27 genera were shared among 95% of samples. The ratio of the families Coriobacteriaceae to Bifidobacteriaceae was significantly different between housing types and farms with pasture fed animals having a higher relative abundance of Coriobacteriaceae . A majority of samples were positive for at least one OTU assigned to Enterobacteriaceae and 31% of samples contained OTUs assigned to Campylobacter . However, the relative abundance of both taxa was <0.1%. The microbial composition displays individual farm specific signatures, but housing type plays a role. These data provide insights into the composition of the core fecal microbiota of commercial dairy cows in California and will further generate hypotheses for strategies to manipulate the microbiome of cattle.

A healthy gastrointestinal (GI) tract with a properly established microbiota is necessary for a foal to develop into a healthy weanling. A foal's health can be critically impacted by aberrations in the microbiome such as with diarrhea which can cause great morbidity and mortality in foals. In this study, we hypothesized that gut establishment in the foal transitioning from a diet of milk to a diet of grain, forage, and pasture would be detectable through analyses of the fecal microbiotas. Fecal samples from 37 sets of foals and mares were collected at multiple time points ranging from birth to weaning. Bacterial DNA was isolated from the samples, and the V4 domain of bacterial 16S rRNA genes were amplified via polymerase chain reaction. Next generation sequencing was then performed on the resulting amplicons, and analyses were performed to characterize the microbiome as well as the relative abundance of microbiota present. We found that bacterial population compositions followed a pattern throughout the early life of the foal in an age-dependent manner. As foals transitioned from milk consumption to a forage and grain diet, there were recognizable changes in fecal microbial compositions from initial populations predominant in the ability to metabolize milk to populations capable of utilizing fibrous plant material. We were also able to recognize differences in microbial populations amongst diarrheic foals as well as microbial population differences associated with differences in management styles between facilities. Future efforts will gauge the effects of lesser abundant bacterial populations that could also be essential to GI health, as well as to determine how associations between microbial population profiles and animal management practices can be used to inform strategies for improving upon the health and growth of horses overall., Competing Interests: The authors have declared that no competing interests exist.

Malnutrition remains a leading contributor to the morbidity and mortality of children under the age of 5 years and can weaken the immune system and increase the severity of concurrent infections. Livestock milk with the protective properties of human milk is a potential therapeutic to modulate intestinal microbiota and improve outcomes. The aim of this study was to develop an infection model of childhood malnutrition in the pig to investigate the clinical, intestinal and microbiota changes associated with malnutrition and enterotoxigenic Escherichia coli (ETEC) infection and to test the ability of goat milk and milk from genetically engineered goats expressing the antimicrobial human lysozyme (hLZ) milk to mitigate these effects. Pigs were weaned onto a protein-energy-restricted diet and after 3 weeks were supplemented daily with goat, hLZ or no milk for a further 2 weeks and then challenged with ETEC. The restricted diet enriched faecal microbiota in Proteobacteria as seen in stunted children. Before infection, hLZ milk supplementation improved barrier function and villous height to a greater extent than goat milk. Both goat and hLZ milk enriched for taxa (Ruminococcaceae) associated with weight gain. Post-ETEC infection, pigs supplemented with hLZ milk weighed more, had improved Z-scores, longer villi and showed more stable bacterial populations during ETEC challenge than both the goat and no milk groups. This model of childhood disease was developed to test the confounding effects of malnutrition and infection and demonstrated the potential use of hLZ goat milk to mitigate the impacts of malnutrition and infection.

Lysozyme is an important non-specific immune protein in human milk, modulating the immune response against bacterial infections. The aim of this study was to characterize the milk of a transgenic goat expressing a recombinant human lysozyme (rhLZ) in the milk, also testing the in vitro antibacterial activity of the rhLZ milk against pathogens of the gastrointestinal tract. Milk samples collected from Tg and non-transgenic goats (nTg) from the 3rd to the 11th week of lactation were submitted to physicochemical analyses, rhLZ semi-quantification, and to rhLZ antimicrobial activity against Micrococcus luteus, Shiguella sonnei and Enterococcus faecalis. Viability and cell migration were studied in ileum epithelial cells (IEC-18) in absence or presence of E. faecalis, Staphylococcus aureus, Escherichia coli (EPEC) and S. sonnei. The expression of ZO-1 and IL-6 genes was evaluated in IEC-18 to evaluate the effect of rhLZ milk on intestinal barrier function and intestinal inflammation. Physicochemical parameters between goat Tg and nTg milk were similar and within normal values for human consumption, with hLZ concentrations being similar between Tg (224μg/mL) and human (226μg/mL) milk. The Tg milk had bactericidal activity against M. luteus, no bactericidal effect on S. sonnei, and relative to discrete sensitivity against E. feacalis than controls. Better migrating parameters were observed in cells in culture with nTg and Tg than controls. In the presence of pathogens, the Tg milk promoted improved migrating parameters than controls, except for S. sonnei, with lower cell numbers in the presence of nTg samples and E. faecalis and S. sonnei. No differences in ZO-1 relative expression patterns were observed in cultured cells, with increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg group being similar to all groups. In conclusion, goat milk containing rhLZ demonstrated valid evidence for its potential use as a nutraceutical for improvement of health and nutrition quality in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Diarrheal diseases in infancy and childhood are responsible for substantial morbidity and mortality in developing nations. Lysozyme, an antimicrobial component of human milk, is thought to play a role in establishing a healthy intestinal microbiota and immune system. Consumption of breast milk has been shown to prevent intestinal infections and is a recommended treatment for infants with diarrhea. Objective: This study aimed to examine the ability of lysozyme-rich goat milk to prevent intestinal infection. Methods: Six-week-old Hampshire-Yorkshire pigs were assigned to treatment groups balanced for weight, sex, and litter and were fed milk from nontransgenic control goats (GM group) or human lysozyme transgenic goats (hLZM group) for 2 wk before they were challenged with porcine-specific enterotoxigenic Escherichia coli (ETEC). Fecal consistency, complete blood counts, intestinal histology, and microbial populations were evaluated. Results: Pigs in the hLZM group had less severe diarrhea than did GM pigs at 24 and 48 h after ETEC infection ( P = 0.01 and 0.05, respectively), indicating a less severe clinical disease state. Relative to baseline, postmilk hLZM pigs had 19.9% and 137% enrichment in fecal Bacteroidetes ( P = 0.028) and Paraprevotellaceae ( P = 0.003), respectively, and a 93.8% reduction in Enterobacteriaceae ( P = 0.007), whereas GM pigs had a 60.9% decrease in Lactobacillales ( P = 0.003) and an 83.3% enrichment in Burkholderiales ( P = 0.010). After ETEC infection, hLZM pigs tended to have lower amounts (68.7% less) of fecal Enterobacteriaceae than did GM pigs ( P = 0.058). There were 83.1% fewer bacteria translocated into the mesenteric lymph nodes of hLZM pigs than into those of GM pigs ( P = 0.039), and hLZM pigs had 34% lower mucin 1 and 61% higher tumor necrosis factor-α expression in the ileum than did GM pigs ( P = 0.046 and 0.034, respectively). Conclusion: Results of this study indicate that human lysozyme milk consumption before and during ETEC infection has a positive effect on clinical disease, intestinal mucosa, and gut microbiota in young pigs., Competing Interests: Author disclosures: LCG, CAC, MWD, J-LW, JDM, and EAM, no conflicts of interest., (© 2017 American Society for Nutrition.)

Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF., Competing Interests: The authors have declared that no competing interests exist.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 70 % 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 500 years. 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.

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.

Avian gastrointestinal (GI) tracts are highly populated with a diverse array of microorganisms that share a symbiotic relationship with their hosts and contribute to the overall health and disease state of the intestinal tract. The microbiome of the young chick is easily prone to alteration in its composition by both exogenous and endogenous factors, especially during the early posthatch period. The genetic background of the host and exposure to pathogens can impact the diversity of the microbial profile that consequently contributes to the disease progression in the host. The objective of this study was to profile the composition and structure of the gut microbiota in young chickens from two genetically distinct highly inbred lines. Furthermore, the effect of the Salmonella Enteritidis infection on altering the composition makeup of the chicken microbiome was evaluated through the 16S rRNA gene sequencing analysis. One-day-old layer chicks were challenged with S. Enteritidis and the host cecal microbiota profile as well as the degree of susceptibility to Salmonella infection was examined at 2 and 7 days post infection. Our result indicated that host genotype had a limited effect on resistance to S. Enteritidis infection. Alpha diversity, beta diversity, and overall microbiota composition were analyzed for four factors: host genotype, age, treatment, and postinfection time points. S. Enteritidis infection in young chicks was found to significantly reduce the overall diversity of the microbiota population with expansion of Enterobacteriaceae family. These changes indicated that Salmonella colonization in the GI tract of the chickens has a direct effect on altering the natural development of the GI microbiota. The impact of S. Enteritidis infection on microbial communities was also more substantial in the late stage of infection. Significant inverse correlation between Enterobacteriaceae and Lachnospiraceae family in both non-infected and infected groups, suggested possible antagonistic interaction between members of these two taxa, which could potentially influences the overall microbial population in the gut. Our results also revealed that genetic difference between two lines had minimal effect on the establishment of microbiota population. Overall, this study provided preliminary insights into the contributing role of S. Enteritidis in influencing the overall makeup of chicken's gut microbiota.

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.

To protect infants from infection, human milk contains high levels of the enzyme lysozyme, unlike the milk of dairy animals. We have genetically engineered goats to express human lysozyme (hLZ milk) in their milk at 68% the amount found in human milk to help extend this protection. This study looked at the effect of hLZ on bacteria in raw milk over time. As the microbial diversity of goats' milk has yet to be investigated in depth using next-generation sequencing (NGS) technologies, we applied NGS and clone library sequencing (CLS) to determine the microbiota of raw goat milk (WT milk) and hLZ milk at early, mid and late lactation. Overall, in WT milk, the bacterial populations in milk at early and mid lactation were similar to each other with a shift occurring at late lactation. Both methods found Proteobacteria as the dominant bacteria at early and mid lactation, while Actinobacteria surged at late lactation. These changes were related to decreases in Pseudomonas and increases in Micrococcus. The bacterial populations in hLZ milk were similar to WT milk at early and mid lactation with the only significant differences occurring at late lactation with the elevation of Bacillaceae, Alicyclobacillaceae, Clostridiaceae and Halomonadaceae., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats., Results: Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection., Conclusions: Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

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.

Risk assessment in transgenic plants is intrinsically different than that for transgenic animals; however both require the verification of proper transgene function and in conjunction, an estimate of any unintended effects caused by expression of the transgene. This work was designed to gather data regarding methodologies to detect pleiotropic effects at the whole animal level using a line of transgenic goats that produce the antimicrobial protein human lysozyme (hLZ) in their milk with the goal of using the milk to treat childhood diarrhea. Metabolomics was used to determine the serum metabolite profile of both the host (lactating does) and non-target organism (kid goats raised on control or hLZ milk) prior to weaning (60 days), at weaning (90 days) and 1 month post-weaning (120 days). In addition, intestinal histology of the kid goats was also carried out. Histological analysis of intestinal segments of the pre-weaning group revealed significantly wider duodenal villi (p = 0.014) and significantly longer villi (p = 0.028) and deeper crypts (p = 0.030) in the ileum of kid goats consuming hLZ milk. Serum metabolomics was capable of detecting differences over time but revealed no significant differences in metabolites between control and hLZ fed kids after correction for false discovery rate. Serum metabolomics of control or hLZ lactating does showed only one significant difference in an unknown metabolite (q = 0.0422). The results as a whole indicate that consumption of hLZ milk results in positive or insignificant intestinal morphology and metabolic changes. This work contributes to the establishment of the safety and durability of the hLZ mammary-specific transgene.

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.

Lactoferrin is an antimicrobial and immunomodulatory protein that is produced in high quantities in human milk and aids in the gastrointestinal (GI) maturation of infants. Beneficial health effects have been observed when supplementing human and animal diets with lactoferrin. A herd of genetically engineered cattle that secrete recombinant human lactoferrin in their milk (rhLF-milk) have been generated which provide an efficient production system and ideal medium for rhLF consumption. The effects of consumption of rhLF-milk were tested on young pigs as an animal model for the GI tract of children. When comparing rhLF-milk fed pigs to non-transgenic milk fed pigs (control), we observed that rhLF-milk fed pigs had beneficial changes in circulating leukocyte populations. There was a significant decrease in neutrophils (p = 0.0036) and increase in lymphocytes (p = 0.0017), leading to a decreased neutrophil to lymphocyte ratio (NLR) (p = 0.0153), which is an indicator of decreased systemic inflammation. We also observed changes in intestinal villi architecture. In the duodenum, rhLF-milk fed pigs tended to have taller villi (p = 0.0914) with significantly deeper crypts (p < 0.0001). In the ileum, pigs consuming rhLF-milk had villi that were significantly taller (p = 0.0002), with deeper crypts (p < 0.0001), and a thinner lamina propria (p = 0.0056). We observed no differences in cytokine expression between rhLF-milk and control-milk fed pigs, indicating that consumption of rhLF-milk did not change cytokine signaling in the intestines. Overall favorable changes in systemic health and GI villi architecture were observed; indicating that consumption of rhLF-milk has the potential to induce positive changes in the GI tract.

The composition of human milk is tailored to contribute to the development of the gastrointestinal (GI) tract of newborns and infants. Importantly, human milk contains the antimicrobial compounds lysozyme and lactoferrin that are thought to contribute to the formation of a health-promoting microbiota. As these protective factors are lacking in the milk of dairy animals, we genetically engineered goats expressing human lysozyme in their milk and have recently reported a new animal model to dissect out the role of milk components on gut microbiota formation. Using the pig as a more human-relevant animal model, we demonstrated that consumption of lysozyme-rich milk enriched the abundance of bacteria associated with GI health and decreased those associated with disease, much like human milk. This work demonstrated that the pig is a valid animal model for gut microbiome studies on the effects of dietary components on microbiota composition, host-microbe interactions and state of the intestine.

Childhood diarrhea is a significant problem in many developing countries and E. coli is a main causative agent of diarrhea in young children. Lysozyme is an antimicrobial protein highly expressed in human milk, but not ruminant milk, and is thought to help protect breastfeeding children against diarrheal diseases. We hypothesized that consumption of milk from transgenic goats which produce human lysozyme (hLZ-milk) in their milk would accelerate recovery from bacterial-induced diarrhea. Young pigs were used as a model for children and infected with enterotoxigenic E. coli. Once clinical signs of diarrhea developed, pigs were fed hLZ-milk or non-transgenic control goat milk three times a day for two days. Clinical observations and complete blood counts (CBC) were performed. Animals were euthanized and samples collected to assess differences in histology, cytokine expression and bacterial translocation into the mesenteric lymph node. Pigs consuming hLZ-milk recovered from clinical signs of infection faster than pigs consuming control milk, with significantly improved fecal consistency (p = 0.0190) and activity level (p = 0.0350). The CBC analysis showed circulating monocytes (p = 0.0413), neutrophils (p = 0.0219), and lymphocytes (p = 0.0222) returned faster to pre-infection proportions in hLZ-milk fed pigs, while control-fed pigs had significantly higher hematocrit (p = 0.027), indicating continuing dehydration. In the ileum, pigs fed hLZ-milk had significantly lower expression of pro-inflammatory cytokine IL-8 (p = 0.0271), longer intestinal villi (p<0.0001), deeper crypts (p = 0.0053), and a thinner lamina propria (p = 0.0004). These data demonstrate that consumption of hLZ-milk helped pigs recover from infection faster, making hLZ-milk an effective treatment of E. coli-induced diarrhea.

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., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Human milk contains antimicrobial factors such as lysozyme and lactoferrin that are thought to contribute to the development of an intestinal microbiota beneficial to host health. However, these factors are lacking in the milk of dairy animals. Here we report the establishment of an animal model to allow the dissection of the role of milk components in gut microbiota modulation and subsequent changes in overall and intestinal health. Using milk from transgenic goats expressing human lysozyme at 68%, the level found in human milk and young pigs as feeding subjects, the fecal microbiota was analyzed over time using 16S rRNA gene sequencing and the G2 Phylochip. The two methods yielded similar results, with the G2 Phylochip giving more comprehensive information by detecting more OTUs. Total community populations remained similar within the feeding groups, and community member diversity was changed significantly upon consumption of lysozyme milk. Levels of Firmicutes (Clostridia) declined whereas those of Bacteroidetes increased over time in response to the consumption of lysozyme-rich milk. The proportions of these major phyla were significantly different (P < 0.05) from the proportions seen with control-fed animals after 14 days of feeding. Within phyla, the abundance of bacteria associated with gut health (Bifidobacteriaceae and Lactobacillaceae) increased and the abundance of those associated with disease (Mycobacteriaceae, Streptococcaceae, Campylobacterales) decreased with consumption of lysozyme milk. This study demonstrated that a single component of the diet with bioactivity changed the gut microbiome composition. Additionally, this model enabled the direct examination of the impact of lysozyme on beneficial microbe enrichment versus detrimental microbe reduction in the gut microbiome community.

Background: Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC., Methods: Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18)., Results: Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC., Conclusions: These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

In addition to its well-recognized antimicrobial properties, lysozyme can also modulate the inflammatory response. This ability may be particularly important in the gastrointestinal tract where inappropriate inflammatory reactions can damage the intestinal epithelium, leading to significant health problems. The consumption of milk from transgenic goats producing human lysozyme (hLZ) in their milk therefore has the potential to positively impact intestinal health. In order to investigate the effect of hLZ-containing milk on the inflammatory response, young pigs were fed pasteurized milk from hLZ or non-transgenic control goats and quantitative real-time PCR was performed to assess local expression of TNF-α, IL-8, and TGF-β1 in the small intestine. Histological changes were also investigated, specifically looking at villi width, length, crypt depth, and lamina propria thickness along with cell counts for intraepithelial lymphocytes and goblet cells. Significantly higher expression of anti-inflammatory cytokine TGF-β1 was seen in the ileum of pigs fed pasteurized milk containing hLZ (P = 0.0478), along with an increase in intraepithelial lymphocytes (P = 0.0255), and decrease in lamina propria thickness in the duodenum (P = 0.0001). Based on these results we conclude that consuming pasteurized milk containing hLZ does not induce an inflammatory response and improves the health of the small intestine in pigs.

While there are many reports in the literature describing the attributes of specific applications of transgenic animals for agriculture, there are relatively few studies focusing on the fitness of the transgenic animals themselves. This work was designed to gather information on genetically modified food animals to determine if the presence of a transgene can impact general animal production traits. More specifically, we used a line of transgenic dairy goats expressing human lysozyme in their mammary gland to evaluate the reproductive fitness and growth and development of these animals compared to their non-transgenic counterparts and the impact of consuming a transgenic food product, lysozyme-containing milk. In males, none of the parameters of semen quality, including semen volume and concentration, total sperm per ejaculate, sperm morphology, viability and motility, were significantly different between transgenic bucks and non-transgenic full-sib controls. Likewise, transgenic females of this line did not significantly differ in the reproductive traits of gestation length and litter size compared to their non-transgenic counterparts. To evaluate growth, transgenic and non-transgenic kid goats received colostrum and milk from either transgenic or non-transgenic does from birth until weaning. Neither the presence of the transgene nor the consumption of milk from transgenic animals significantly affected birth weight, weaning weight, overall gain and post-wean gain. These results indicate that the analyzed reproductive and growth traits were not regularly or substantially impacted by the presence or expression of the transgene. The evaluation of these general parameters is an important aspect of defining the safety of applying transgenic technology to animal agriculture.

Nutrition, bacterial composition of the gastrointestinal tract, and general health status can all influence the metabolic profile of an organism. We previously demonstrated that feeding pasteurized transgenic goats' milk expressing human lysozyme (hLZ) can positively impact intestinal morphology and modulate intestinal microbiota composition in young pigs. The objective of this study was to further examine the effect of consuming hLZ-containing milk on young pigs by profiling serum metabolites. Pigs were placed into two groups and fed a diet of solid food and either control (non-transgenic) goats' milk or milk from hLZ-transgenic goats for 6 weeks. Serum samples were collected at the end of the feeding period and global metabolite profiling was performed. For a total of 225 metabolites (160 known, 65 unknown) semi-quantitative data was obtained. Levels of 18 known and 4 unknown metabolites differed significantly between the two groups with the direction of change in 13 of the 18 known metabolites being almost entirely congruent with improved health status, particularly in terms of the gastrointestinal tract health and immune response, with the effects of the other five being neutral or unknown. These results further support our hypothesis that consumption of hLZ-containing milk is beneficial to health.

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.

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.

The insertion of foreign DNA at a specific genomic locus directed by homologous DNA sequences, or gene targeting, is an inefficient process in mammalian somatic cells. Given the key role of non-homologous end joining (NHEJ) pathway in DNA double-strand break (DSB) repair in mammalian cells, we investigated the effects of decreasing NHEJ protein levels on gene targeting. Here we demonstrate that the transient knockdown of integral NHEJ proteins, Ku70 and Xrcc4, by RNAi in human HCT116 cells has a remarkable effect on gene targeting/random insertions ratios. A timely transfection of an HPRT-based targeting vector after RNAi treatment led to a 70% reduction in random integration events and a 33-fold increase in gene targeting at the HPRT locus. These findings bolster the role of NHEJ proteins in foreign DNA integration in vivo, and demonstrate that their transient depletion by RNAi is a viable approach to increase the frequency of gene targeting events. Understanding how foreign DNA integrates into a cell's genome is important to advance strategies for biotechnology and genetic medicine.

Transgenesis provides a method of expressing novel proteins in milk to increase the functional benefits of milk consumption. Transgenic goats expressing human lysozyme (hLZ) at 67% of the concentration in human breast milk were produced, thereby enhancing the antimicrobial properties of goats' milk. The objective of this study was to investigate the impact of pasteurized milk containing hLZ on growth, the intestinal epithelium, and an enteropathogenic Escherichia coli (EPEC) infection in young weaned pigs. Pigs were placed into 4 groups and fed a diet of solid food and either control (nontransgenic) goats' milk or milk from hLZ-transgenic goats. Growth was assessed by weight gain. Nonchallenged pigs were necropsied after 6 wk, whereas the remaining pigs were necropsied at 7 wk following bacterial challenge. We determined the numbers of total coliforms and E. coli and examined small intestinal histology for all pigs. Complete blood counts were also determined pre- and postchallenge. Challenged pigs receiving hLZ milk had fewer total coliforms (P = 0.029) and E. coli (P = 0.030) in the ileum than controls. hLZ-fed pigs also had a greater duodenal villi width (P = 0.029) than controls. Additionally, nonchallenged hLZ-fed pigs had fewer intraepithelial lymphocytes per micron of villi height (P = 0.020) than nonchallenged controls. These results indicate that the consumption of pasteurized hLZ goats' milk has the potential to improve gastrointestinal health and is protective against an EPEC in young weaned pigs. These same benefits may occur in young children if they were to consume milk from hLZ-transgenic goats.

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.

Non-homologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells and is likely responsible for the non-homologous integration of transgenes. In higher eukaryotes, this pathway predominates over the homologous recombination (HR) pathway and therefore may account for the low level of HR events that occur in mammalian cells. We evaluated the effects of transient RNAi-induced down-regulation of key components of the NHEJ pathway in human HCT116 cells. Treatment with siRNA targeting Ku70 and Xrcc4 reduced corresponding protein levels by 80-90% 48h after transfection, with a return to normal levels by 96h. Additionally, down-regulation of Ku70 and Xrcc4 resulted in a concomitant depletion of both Ku70 and Ku86 proteins. Biological consequences of transient RNAi-mediated depletion of Ku70 and Xrcc4 included sensitization to gamma radiation and a significant decrease in the expression of a linear GFP reporter gene. The results highlight the possibility of a successful means to manipulate the NHEJ pathway by RNAi.

Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb alpha(S1)-casein/human lysozyme DNA construct and a small 5.5 kb beta-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born.

The addition of human milk components with intrinsic antimicrobial activity to livestock milk by genetic engineering has the potential to benefit milk safety and production as well as the health of the lactating animal. As a model for the dairy cow, we generated transgenic goats that expressed human lysozyme in their milk at 68% of the levels found in human milk. Milk from these transgenic animals had a bacteriostatic effect on both in vitro and in vivo growth of several microorganisms important to the dairy industry. In vitro, milk from transgenic animals was capable of slowing the growth of mastitis-causing strains of Escherichia coli (P < 0.02) and Staphylococcus aureus (P < 0.05) as well as the cold-spoilage organism Pseudomonas fragi (P < 0.02). The growth of an organism involved in cheese-making, Lactococcus lactis, was not affected by the presence of lysozyme in milk. The supplementation of control milk with purified lysozyme did not achieve the same inhibitory effect as milk from transgenic animals. In vivo, milk from transgenic animals supported less bacterial growth than control milk. This transgenic model demonstrates the possibilities offered by genetic engineering to enhance the antimicrobial nature of milk and the udder.