Shanghai Transgenic Research Center

Shanghai, China

Shanghai Transgenic Research Center

Shanghai, China
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Yu H.,Shanghai JiaoTong University | Yu H.,Shanghai Transgenic Research Center | Chen J.,Shanghai Transgenic Research Center | Sun W.,Shanghai Transgenic Research Center | And 8 more authors.
Journal of Biotechnology | Year: 2012

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3. kb hLF minigene to the regulatory elements of the β-casein gene. The transgenic goat produced more than 30. mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future. © 2012.


Yu H.,Shanghai JiaoTong University | Yu H.,Shanghai Transgenic Research Center | Wang X.,Shanghai Transgenic Research Center | Zhu L.,Shanghai Transgenic Research Center | And 6 more authors.
Gene | Year: 2013

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site. © 2012 Elsevier B.V.


Bao Z.,Nanjing Agricultural University | Gao X.,Nanjing Agricultural University | Zhang Q.,Nanjing Agricultural University | Lin J.,Nanjing Agricultural University | And 5 more authors.
PLoS ONE | Year: 2015

The development of genetically engineered animals has brought with it increasing concerns about biosafety issues. We therefore evaluated the risks of growth hormone from transgenic goats, including the probability of horizontal gene transfer and the impact on the microbial community of the goats' gastrointestinal tracts, feces and the surrounding soil. The results showed that neither the GH nor the neoR gene could be detected in the samples. Moreover, there was no significant change in the microbial community of the gastrointestinal tracts, feces and soil, as tested with PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing. Finally, phylogenetic analysis showed that the intestinal content, feces and soil samples all contained the same dominant group of bacteria. These results demonstrated that expression of goat growth hormone in the mammary of GH transgenic goat does not influence the microflora of the intestine, feces and surrounding soil. © 2015 Bao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Fan J.-H.,Huangpu District Center Hospital | Cheng G.-X.,Shanghai Transgenic Research Center
Chinese Journal of Cancer Prevention and Treatment | Year: 2012

OBJECTIVE: To prepare of EGFRv III Monoclonal Antibody, and it is expected to be good to the tumor's target treatment. METHODS: The peptide of EGFRv III was synthesized, and conjugated with KLH. Balb/c mouse was immuned, hybridoma cell was used to generate ascites, antibody from ascites was purified and the speciality of the monoclonal antibody was determined. RESULTS: The corresponding 14-amino acid peptide of the gene absence fusing area of EGFRv III, and conjugatd with KLH. Immune Balb/c mouse using this conjugation as antigen with a certain immune protocal, when high titer antiserum (1:128 000) was obtained, fuse the spleen cell of immuned mouse with SP2/0 cell, select by ELISA and subclone to obtain anti EGFRv III stabilized hybridoma cell lines. 5 mouse hybridoma lines obtained and ascites produced. The type of Monoclonal Antibody is IgG2a, Determine ascites antibody titer and it's affinity with antigen. The titer was 1:128000, and the highest affinity of those was 9.8×10 -9 mol/L. The speciality of the monoclonal antibody was determined by the expressed natural EGFR's binding domain in Pichia yeast. The results showed that the antibody could not bind with the expressed peptide. It indicated that the monoclonal antibody prepared by ourselves was special to EGFRv III. CONCLUSION: The succeeding preparation of EGFRv III monoclonal antibody by this experiment gives a stability base to the tumor's target treatment.


Yao L.,Chongqing Medical University | Wang P.,Fudan University | Liu J.,Chongqing Medical University | Chen J.,Shanghai Transgenic Research Center | And 2 more authors.
International Journal of Clinical and Experimental Medicine | Year: 2014

Poor development of the interspecies somatic cell nuclear transfer (iSCNT) embryos was due to nuclear-mitochondrial incompatibility. In humans, it has been known that ooplast transfer (OT) could support normal fertilization, the development of embryos and prevents the transmission of mtDNA disease. To investigate whether OT could support development of the iSCNT embryos, the ooplast of Triploid Pronucleus (3PN) zygote which would be discarded in IVF lab was transferred into the enucleated goat oocytes to construct humanized iSCNT embryos in our study. The results showed the 3PN-OT could significantly improve the early development of humanized iSCNT embryos. The percentage of blastocyst development of OT group was also higher than that of the control group. Interestingly, the morphology of some OT-iSCNT blastocysts was similar to normal human blastocysts in vitro fertilization, while the morphology of iSCNT blastocysts from control group was similar to goat blastocysts. Importantly, the pluripotent marker Oct4 of the OT-iSCNT blastocyst was expressed stronger than that of the control group. These results suggested that 3PN-OT could improve the developmental potency of human iSCNT embryos and would facilitate establishing ESCs from iSCNT blastocysts. © 2014 E-Century Publishing Corporation. All rights reserved.


Zhang Q.,Nanjing Agricultural University | Chen J.Q.,Shanghai Transgenic Research Center | Lin J.,Nanjing Agricultural University | Yu Q.H.,Nanjing Agricultural University | And 4 more authors.
Molecular Biology Reports | Year: 2014

Growth hormone is a positive regulator of mammary gland development. Dairy animals that are administered growth hormone display enhanced lactation performance, a desirable agricultural trait. The objective of the current research was to generate an improved milk production phenotype in a large animal model using over-expressed GH in the mammary gland to promote mammogenesis. To this end, we constructed a mammary gland-specific expression vector, pcGH, and demonstrated effective GH expression in goat mammary epithelial cells in vitro by ELISA. Then, to produce transgenic offspring that were capable of stable GH expression in vivo, the linearized pcGH vector was electroporated into goat fetal fibroblasts. Cell colonies that were positive for GH were used as donors for nuclear transfer to enucleated oocytes. A total of 253 morulae or blastocytes developed from the reconstructed embryos were transferred to 56 recipients, resulting in 24 pregnancies at day 35. Finally, six transgenic goats were born. PCR detection confirmed the success of the cloning procedure. To observe the mammogenesis of dairy goats, the GH transgenic goats were mated with a completely healthy buck. In the later pregnancy period, the mammary gland of the GH transgenic goats were extensive than non-transgenic goats. These experiments indicated that the pcGH vector was incorporated into the transgenic goats and affected mammogenesis, which laid a solid foundation for elucidating the impact of GH on mammogenesis and lactation performance. © 2014 Springer Science+Business Media.


PubMed | Shanghai Transgenic Research Center and Nanjing Agricultural University
Type: Journal Article | Journal: PloS one | Year: 2015

The development of genetically engineered animals has brought with it increasing concerns about biosafety issues. We therefore evaluated the risks of growth hormone from transgenic goats, including the probability of horizontal gene transfer and the impact on the microbial community of the goats gastrointestinal tracts, feces and the surrounding soil. The results showed that neither the GH nor the neoR gene could be detected in the samples. Moreover, there was no significant change in the microbial community of the gastrointestinal tracts, feces and soil, as tested with PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing. Finally, phylogenetic analysis showed that the intestinal content, feces and soil samples all contained the same dominant group of bacteria. These results demonstrated that expression of goat growth hormone in the mammary of GH transgenic goat does not influence the microflora of the intestine, feces and surrounding soil.


Patent
Shanghai Genon Bioengineering Co. and Shanghai Transgenic Research Center | Date: 2016-08-03

Provided in the present invention is a method for positioning and integrating transgene and a use thereof. Specifically provided is a variant loxP element. The sequence ATAAT of an reverse repeated sequence in a wild type loxp locus is mutated into CACCT, i.e., a variant loxp element. Also provided in the present invention are a construct comprising the variant loxP element, a vector or host cell comprising the construct and a method for preparing a transgenic animal using the vector and the host cell.


Patent
Shanghai Genon Bioengineering Co. and Shanghai Transgenic Research Center | Date: 2013-09-28

Provided in the present invention is a method for positioning and integrating transgene and a use thereof. Specifically provided is a variant loxP element. The sequence ATAAT of an reverse repeated sequence in a wild type loxp locus is mutated into CACCT, i.e., a variant loxp element. Also provided in the present invention are a construct comprising the variant loxP element, a vector or host cell comprising the construct and a method for preparing a transgenic animal using the vector and the host cell.


PubMed | Shanghai Transgenic Research Center
Type: | Journal: Journal of biotechnology | Year: 2014

Human lysozyme (hLZ), an essential protein against many types of microorganisms, has been expressed in transgenic livestock to improve their health status and milk quality. However, the large-scale production of hLZ in transgenic livestock is currently unavailable. Here we describe the generation of transgenic goats, by somatic cell-mediated transgenic cloning, that express large amounts of recombinant human lysozyme (rhLZ) in milk. Specifically, two optimized lysozyme expression cassettes (-casein/hLZ and -lactoglobulin/hLZ) were designed and introduced into goat somatic cells by cell transfection. Using transgenic cell colonies, which were screened by 0.8mg/ml G418, as a nuclear donor, we obtained 10 transgenic cloned goats containing one copy of hLZ hybrid gene. An ELISA assay indicated that the transgenic goats secreted up to 6.2g/L of rhLZ in their milk during the natural lactation period, which is approximately 5-10 times higher than human milk. The average rhLZ expression levels in -casein/hLZ and -lactoglobulin/hLZ transgenic goats were 2.3g/L and 3.6g/L, respectively. Therefore, both rhLZ expression cassettes could induce high levels of expression of the rhLZ in goat mammary glands. In addition, the rhLZ purified from goat milk has similar physicochemical properties as the natural human lysozyme, including the molecular mass, N-terminal sequence, lytic activity, and thermal and pH stability. An antibacterial analysis revealed that rhLZ and hLZ were equally effective in two bacterial inhibition experiments using Staphylococcus aureus and Escherichia coli. Taken together, our experiments not only underlined that the large-scale production of biologically active rhLZ in animal mammary gland is realistic, but also demonstrated that rhLZ purified from goat milk will be potentially useful in biopharmaceuticals.

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