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Sui S.,China Agricultural University | Zhao J.,China Agricultural University | Wang J.,Beijing GenProtein Biotech Co. | Wang J.,Wuxi Kingenew Biotechnology Co. | And 4 more authors.

The use of transgenic livestock is providing new methods for obtaining pharmaceutically useful proteins. However, the protein expression profiles of the transgenic animals, including expression of milk fat globule membrane (MFGM) proteins, have not been well characterized. In this study, we compared the MFGM protein expression profile of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle, i.e., expressing recombinant human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland, with those from cloned non-transgenic (C) and conventionally bred normal animals (N). We identified 1, 225 proteins in milk MFGM, 166 of which were specifically expressed only in the TC-LA group, 265 only in the TC-LF group, and 184 only in the TC-LZ group. There were 43 proteins expressed only in the transgenic cloned animals, but the concentrations of these proteins were below the detection limit of silver staining. Functional analysis also showed that the 43 proteins had no obvious influence on the bovine mammary gland. Quantitative comparison revealed that MFGM proteins were up- or down-regulated more than twofold in the TC and C groups compared to N group: 126 in colostrum and 77 in mature milk of the TC-LA group; 157 in colostrum and 222 in mature milk of the TC-LF group; 49 in colostrum and 98 in mature milk of the TC-LZ group; 98 in colostrum and 132 in mature milk in the C group. These up- and down-regulated proteins in the transgenic animals were not associated with a particular biological function or pathway, which appears that expression of certain exogenous proteins has no general deleterious effects on the cattle mammary gland. © 2014 Sui et al. Source

Zhang R.,China Agricultural University | Guo C.,China Agricultural University | Sui S.,China Agricultural University | Yu T.,Wuxi Kingenew Biotechnology Co. | And 2 more authors.

The development of transgenic cloned animals offers new opportunities for agriculture, biomedicine and environmental science. Expressing recombinant proteins in dairy animals to alter their milk composition is considered beneficial for human health. However, relatively little is known about the expression profile of the proteins in milk derived from transgenic cloned animals. In this study, we compared the proteome and nutrient composition of the colostrum and mature milk from three lines of transgenic cloned (TC) cattle that specifically express human α-lactalbumin (TC-LA), lactoferrin (TC-LF) or lysozyme (TC-LZ) in the mammary gland with those from cloned non-transgenic (C) and conventionally bred normal animals (N). Protein expression profile identification was performed, 37 proteins were specifically expressed in the TC animals and 70 protein spots that were classified as 22 proteins with significantly altered expression levels in the TC and C groups compared to N group. Assessment of the relationship of the transgene effect and normal variability in the milk protein profiles in each group indicated that the variation in the endogenous protein profiles of the three TC groups was within the limit of natural variability. More than 50 parameters for the colostrum and mature milk were compared between each TC group and the N controls. The data revealed essentially similar profiles for all groups. This comprehensive study demonstrated that in TC cattle the mean values for the measured milk parameters were all within the normal range, suggesting that the expression of a transgene does not affect the composition of milk. © 2012 Zhang et al. Source

Chen X.,China Agricultural University | Wang J.,Beijing GenProtein Biotech Co. | Wang J.,CAS Institute of Zoology | Li R.,Beijing GenProtein Biotech Co. | And 5 more authors.
Cellular Reprogramming

This study was conducted to determine the effect of microinjection of a single blastomere from in vitro fertilization (IVF)-derived eight-cell embryo into eight-cell cloned embryos harboring the gene encoding recombinant human lactoferrin (rhLF), GFP, and NEO markers in bovine. The reconstructed chimeric embryos were assessed for their development to blastocyst, or to term after transfer, and tissues of offspring were evaluated by polymerase chain reaction (PCR) for the presence of nuclear transfer (NT)-derived transgenic cells, and the cloned embryos without microinjection were used as controls. The chimeric embryos showed slightly higher blastocyst rate than that for controls. The single IVF-derived blastomere appeared to preferential contribute to inner cell mass (ICM) in the chiemric blastocysts. After transfer, the rates of development of chimeric embryos to day 60, to term, and to weaning were significantly higher than those of controls. Sixty-three chimeric blastocysts were transferred and 11 calves were born: 7 calves of them were dead, and the remaining 4 calves are apparently normal and healthy. Most of the tissues collected from dead fetus were transgenic, whereas NT-derived transgenic cells were not detected in some tissues of the living calves. Our results indicated that a single blastomere from IVF-derived eight-cell embryo improves the in vivo developmental potential of transgenic cloned eight-cell embryos in bovine; however, the single IVF-derived blastomere appeares to be better able to populate the ICM and many tissues of offspring than NT-derived blastomeres. © 2010 Mary Ann Liebert, Inc. Source

Zhang R.,China Agricultural University | Yin Y.,Life Technologies | Zhang Y.,Life Technologies | Li K.,Life Technologies | And 5 more authors.

As the number of transgenic livestock increases, reliable detection and molecular characterization of transgene integration sites and copy number are crucial not only for interpreting the relationship between the integration site and the specific phenotype but also for commercial and economic demands. However, the ability of conventional PCR techniques to detect incomplete and multiple integration events is limited, making it technically challenging to characterize transgenes. Next-generation sequencing has enabled cost-effective, routine and widespread high-throughput genomic analysis. Here, we demonstrate the use of next-generation sequencing to extensively characterize cattle harboring a 150-kb human lactoferrin transgene that was initially analyzed by chromosome walking without success. Using this approach, the sites upstream and downstream of the target gene integration site in the host genome were identified at the single nucleotide level. The sequencing result was verified by event-specific PCR for the integration sites and FISH for the chromosomal location. Sequencing depth analysis revealed that multiple copies of the incomplete target gene and the vector backbone were present in the host genome. Upon integration, complex recombination was also observed between the target gene and the vector backbone. These findings indicate that next-generation sequencing is a reliable and accurate approach for the molecular characterization of the transgene sequence, integration sites and copy number in transgenic species. © 2012 Zhang et al. Source

Yu T.,China Agricultural University | Guo C.,China Agricultural University | Wang J.,China Agricultural University | Wang J.,CAS Institute of Zoology | And 10 more authors.

The glycosylation profile of a recombinant protein is important because glycan moieties can play a significant role in the biological properties of the glycoprotein. Here we determined the site-specific N-glycosylation profile of human lactoferrin (hLF) and recombinant human lactoferrin (rhLF) expressed in the milk of transgenic cloned cattle. We used combined approaches of monosaccharide composition analysis, lectin blot, glycan permethylation and sequential exoglycosidase digestion and analyzed samples using high-performance ion chromatography and mass spectrometry (MS). Nglycans from hLF are comprised entirely of highly branched, highly sialylated and highly fucosylated complextype structures, and many contain Lewisx epitopes. Six of these structures are reported here for the first time. However, N-glycans from rhLF are of the high mannose-, hybrid- and complex-type structures, with less N-acetylneuraminic acid and fucose. Some contain a terminal Nacetylgalactosamine- N-acetylglucosamine (LacdiNAc) disaccharide sequence. Monosaccharide composition analysis of rhLF revealed small amounts of N-glycolylneuraminic acid, which were not detected by MS. hLF and rhLF appear to be glycosylated at the same two sites: Asn138 and Asn479. The third putative glycosylation site, at Asn624, is unglycosylated in both hLF and rhLF. The relative abundance of each N-glycan at each site was also determined. The different N-glycosylation profile of rhLF when compared with that of hLF is in consistent with the widely held view that glycosylation is species- and tissue/cell-specific. These data provide an important foundation for further studies of glycan structure/function relationships for hLF and rhLF and help to better understand the glycosylation echanism in bovine mammary epithelial cells. © The Author 2010. Published by Oxford University Press. Source

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