Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens.

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.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian Semi Arid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian Semi Arid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.(AU)

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA) / Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Dicing to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80o C. Alternatively, chemically synthesiz

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (1

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNaiTM). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80ºC. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAiTM was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA) / Efficient RNAi-induced protein knockdown in somatic cells using diced or chemically produced small interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Dicing to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80o C. Alternatively, chemically synthesiz

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specifi c corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specifi c mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most effi cient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specifi c and unique siRNA sequences (Stealth RNaiTM).Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (1

Genetic engineering of livestock to improve human health: The human lysozyme transgenic goat model

Background: Transgenic animals have been generated for a variety of purposes including research tools, medical models, bioreactors (dairy animals producing human pharmaceuticals in their milk or in the egg whites of chickens) and for production agriculture (animals with increased growth, decreased environmental pollution, disease resistance), which includes the generation of animals designed to benefit human health. For example, transgenic goats expressing human lysozyme in their milk are being used as a model method to supply milk with antibacterial properties to help fight diarrheal illnesses in children. Lysozyme is a naturally occurring antimicrobial found in human milk at much higher levels than in the milk of dairy goats and cows. Lysozyme serves as part of the natural defense system against infection and also helps establish a healthy gut microbiota in the infant. We hypothesized that the presence of increased levels of lysozyme in the milk of dairy goats could offer several benefits that affect human health, including the promotion of a healthy gut microbiota and associated benefits such as improved growth and resistance to intestinal infections. Review: Research with this line of transgenic goats over the last 12 years has demonstrated that the presence and expression of the human lysozyme transgene is not detrimental to the animals themselves and that the milk can indeed act in an antimicrobial fashion when consumed by pigs, a model animal for human health, and impact the state of the intestine in a positive manner. Pigs consuming milk from human lysozyme transgenic goats had significantly lower levels of coliforms and E. coli in their intestine than did pigs consuming milk from non-transgenic control animals. In addition to bacterial changes, intestinal tissue of pigs consuming milk from lysozyme transgenic goats had a significantly larger surface area with significantly fewer intraepithelial lymphocytes and an elevated level of expression of the anti-inflammatory cytokine TGF-â1 compared to control-fed animals, all indicators of a healthier intestinal tract. Metabolite profile analysis demonstrated significant differences in the levels of 18 metabolites in the serum of pigs fed lysozyme milk with the direction of changes beneficial to the health of the animal. Finally, pigs consuming milk from lysozyme transgenic goats were more resistant to infection when challenged with an enteropathogenic E. coli, indicating a protective effect of lysozyme milk. In the semi-arid northeast of Brazil, 89 of 1000 children die before they reach the age of 2 years and 17% of these deaths can be attributed to diarrhea. The use of genetically engineered animals containing increased levels of lysozyme in their milk is a novel and simple approach to fight this problem. Work will be presented outlining the characterization of these animals and the impact of consuming the milk with the goal of one day having the milk available as a preventative or treatment agent for diarrheal illnesses. Conclusions: Genetic engineering is a viable approach to produce animal food products that can be used to improve human health. Brazil's acceptance of this technology has positioned itself at the forefront to demonstrate to the world a new tool to help fight common diarrhea and its impact on the growth and development of children.

Current status of transgenic animal research for human health applications

Background: The current status of genetic engineering animals for biomedical and human health applications, including improving the supply of animal-based food products, was reviewed. Although transgenic animals have been available for almost 30 years only one product, a drug derived from transgenic goats' milk, has been approved for use anywhere in the world. While a number of technical issues limited efficiency initially, products coming to market were hindered by the lack of a regulatory framework and compounded by opposition from anti-biotechnology groups. Review: As presented in the review, the production of genetically engineered livestock has progressed from the initial technology of pronuclear microinjection through to the wide-spread use today of somatic cell nuclear transfer-based cloning technology following the transfection of cells in culture with methods such as lipofection or electroporation. There also was significant progress in the development of systems based upon lentivirus or adeno-associated virus-based vectors. More recently, advances occurred based on transposon-mediated transgenesis. In conjunction with advances in methods to genetically engineer livestock, there are also advances, such as the use of zinc-finger nucleases and transient depletion of endogenous non-homologous recombination systems, to increase the efficiency of homologous recombination-based gene targeting. Applications range from the production of pharmaceutical proteins, silk for use in sutures and scaffolds for cellular regeneration, and xenotransplantation through to the development of transgenic animals that improve animal production and the nutritional value of animal-based food products. Both of these agricultural applications are important for maintaining the global production of sufficient meat-based products in an economically and environmentally sustainable manner in the face of a dramatically increasing human population. Conclusion: Advances in the production of genetically engineered animals and identification of useful applications have significantly improved and have set the stage for the adoption of transgenic animals. Brazil, because of recent technological developments and the adoption of regulatory guidelines is now in a strong position to benefit from genetically engineered animals and to contribute to the leadership in the adoption of transgenic animals for applications to improve human and animal health and well-being.