The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Yong Cheng - One of the best experts on this subject based on the ideXlab platform.
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the Goat β casein cmv chimeric promoter drives the expression of hlf in Transgenic Goats produced by cell transgene microinjection
International Journal of Molecular Medicine, 2019Co-Authors: Ting Zhang, Minya Zhou, Yuguo Yuan, Tingting Yuan, Kunning Yan, Yong ChengAbstract:There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce Transgenic cloned Goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing Goat β-casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25-rhLF-Neo vector was microinjected into Goat fetal fibroblasts. G418 selection and PCR analysis were used to identify Transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, Goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this Transgenic Goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high-purity (95.8%) rhLF from a cation-exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a Transgenic Goat harboring the hLF transgene driven by the Goat β-casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.
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The Goat β‑casein/CMV chimeric promoter drives the expression of hLF in Transgenic Goats produced by cell transgene microinjection.
International journal of molecular medicine, 2019Co-Authors: Ting Zhang, Minya Zhou, Yuguo Yuan, Tingting Yuan, Kunning Yan, Yong ChengAbstract:There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce Transgenic cloned Goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing Goat β-casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25-rhLF-Neo vector was microinjected into Goat fetal fibroblasts. G418 selection and PCR analysis were used to identify Transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, Goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this Transgenic Goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high-purity (95.8%) rhLF from a cation-exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a Transgenic Goat harboring the hLF transgene driven by the Goat β-casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.
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A Novel Recombinant Human Plasminogen Activator: Efficient Expression and Hereditary Stability in Transgenic Goats and in Vitro Thrombolytic Bioactivity in the Milk of Transgenic Goats
PloS one, 2018Co-Authors: Ting Zhang, Lei Jiang, Minya Zhou, Yong ChengAbstract:Background Thromboses is a rapidly growing medical problem worldwide. Low-cost, high-scale production of thrombotic drugs is needed to meet the demand. The production of biomolecules in Transgenic animals might help address this issue. To our knowledge, the expression of recombinant human plasminogen activator (rhPA) in Goat mammary glands has never been reported before. Methods We constructed a mammary gland–specific expression vector, BLC14/rhPA, which encodes only the essential K2 fibrin-binding and P domains of wild-type tPA (deletion mutant of tPA lacking the F, E, and K1 domains), along with the Goat β-lactoglobulin gene signal peptide-coding sequence. The mammary gland–specific expression vector BLC14/rhPA was transfected into Goat fetal fibroblast cells by electroporation. After selection for 3 weeks by G418, stably transfected cell colonies were obtained. PCR analysis results indicated that 24 of the resistant clones were Transgenic cell lines; of these, 8 lines were selected as the donor cells. The positive cells were starved for 72 h with DMEM/F12 medium containing 0.5% FBS and were then used as do. Finally, 256 reconstructed oocytes were transferred into 26 recipients, and 7 of them became pregnant (pregnancy rate, 26.9%). Two kids were obtained (BP21 and BP22). PCR analysis confirmed that both were Transgenic Goats. To analyze the heredity of the rhPA expressed in BP21 F0 and F1 Transgenic Goats, the F0 Transgenic Goat BP21 was mated with a normal male Goat to generate an F1 Transgenic Goat. Enucleated metaphase II (MII) oocytes and positive donor cells were used to reconstruct embryos, which were transplanted into the oviducts of the recipients. Results Western blot results showed a specific 39 kDa band. The rhPA expression level in Transgenic Goat whey was about 78.32 μg/mL by ELISA. Results of ELISA and the in vitro thrombolysis test (FAPA) showed that specific activity of the rhPA in the milk of F0 and F1 Transgenic Goats was 13.3 times higher than that of the reteplase reference material. Conclusion Thus, we demonstrated that BLC14/rhPA was reasonably effective for expression in the mammary glands of Transgenic Goats, and was stably inherited by the offspring. This study provides the basis for the large-scale production of biological pharmaceuticals in Transgenic animals. The expression of biopharmaceuticals by Transgenic animals can be used for pharmacological research and bioactive analysis, and Transgenic Goats were demonstrated to be promising animals for the large-scale production of thrombolytic biopharmaceuticals.
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ELISA of milk whey from the Transgenic Goat BP21 and F1 Transgenic Goat.
2018Co-Authors: Ting Zhang, Lei Jiang, Minya Zhou, Yong ChengAbstract:ELISA of milk whey from the Transgenic Goat BP21 and F1 Transgenic Goat.
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FAPA analysis of partial whey from Transgenic Goat milk.
2018Co-Authors: Ting Zhang, Lei Jiang, Minya Zhou, Yong ChengAbstract:1–9: Reteplase standard (concentrations are 2,000, 1,500, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μg/mL, respectively); 10–11: whey collected on different days from the Transgenic Goat BP21 (diluted 10-folds); 12: whey collected on different days from the F1 Transgenic Goat (diluted 10-folds); 13, 14: whey from non-Transgenic Goats as the negative control; 15: PBS as the negative control.
Ting Zhang - One of the best experts on this subject based on the ideXlab platform.
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The Goat β‑casein/CMV chimeric promoter drives the expression of hLF in Transgenic Goats produced by cell transgene microinjection.
International journal of molecular medicine, 2019Co-Authors: Ting Zhang, Minya Zhou, Yuguo Yuan, Tingting Yuan, Kunning Yan, Yong ChengAbstract:There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce Transgenic cloned Goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing Goat β-casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25-rhLF-Neo vector was microinjected into Goat fetal fibroblasts. G418 selection and PCR analysis were used to identify Transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, Goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this Transgenic Goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high-purity (95.8%) rhLF from a cation-exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a Transgenic Goat harboring the hLF transgene driven by the Goat β-casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.
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the Goat β casein cmv chimeric promoter drives the expression of hlf in Transgenic Goats produced by cell transgene microinjection
International Journal of Molecular Medicine, 2019Co-Authors: Ting Zhang, Minya Zhou, Yuguo Yuan, Tingting Yuan, Kunning Yan, Yong ChengAbstract:There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce Transgenic cloned Goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing Goat β-casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25-rhLF-Neo vector was microinjected into Goat fetal fibroblasts. G418 selection and PCR analysis were used to identify Transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, Goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this Transgenic Goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high-purity (95.8%) rhLF from a cation-exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a Transgenic Goat harboring the hLF transgene driven by the Goat β-casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.
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A Novel Recombinant Human Plasminogen Activator: Efficient Expression and Hereditary Stability in Transgenic Goats and in Vitro Thrombolytic Bioactivity in the Milk of Transgenic Goats
PloS one, 2018Co-Authors: Ting Zhang, Lei Jiang, Minya Zhou, Yong ChengAbstract:Background Thromboses is a rapidly growing medical problem worldwide. Low-cost, high-scale production of thrombotic drugs is needed to meet the demand. The production of biomolecules in Transgenic animals might help address this issue. To our knowledge, the expression of recombinant human plasminogen activator (rhPA) in Goat mammary glands has never been reported before. Methods We constructed a mammary gland–specific expression vector, BLC14/rhPA, which encodes only the essential K2 fibrin-binding and P domains of wild-type tPA (deletion mutant of tPA lacking the F, E, and K1 domains), along with the Goat β-lactoglobulin gene signal peptide-coding sequence. The mammary gland–specific expression vector BLC14/rhPA was transfected into Goat fetal fibroblast cells by electroporation. After selection for 3 weeks by G418, stably transfected cell colonies were obtained. PCR analysis results indicated that 24 of the resistant clones were Transgenic cell lines; of these, 8 lines were selected as the donor cells. The positive cells were starved for 72 h with DMEM/F12 medium containing 0.5% FBS and were then used as do. Finally, 256 reconstructed oocytes were transferred into 26 recipients, and 7 of them became pregnant (pregnancy rate, 26.9%). Two kids were obtained (BP21 and BP22). PCR analysis confirmed that both were Transgenic Goats. To analyze the heredity of the rhPA expressed in BP21 F0 and F1 Transgenic Goats, the F0 Transgenic Goat BP21 was mated with a normal male Goat to generate an F1 Transgenic Goat. Enucleated metaphase II (MII) oocytes and positive donor cells were used to reconstruct embryos, which were transplanted into the oviducts of the recipients. Results Western blot results showed a specific 39 kDa band. The rhPA expression level in Transgenic Goat whey was about 78.32 μg/mL by ELISA. Results of ELISA and the in vitro thrombolysis test (FAPA) showed that specific activity of the rhPA in the milk of F0 and F1 Transgenic Goats was 13.3 times higher than that of the reteplase reference material. Conclusion Thus, we demonstrated that BLC14/rhPA was reasonably effective for expression in the mammary glands of Transgenic Goats, and was stably inherited by the offspring. This study provides the basis for the large-scale production of biological pharmaceuticals in Transgenic animals. The expression of biopharmaceuticals by Transgenic animals can be used for pharmacological research and bioactive analysis, and Transgenic Goats were demonstrated to be promising animals for the large-scale production of thrombolytic biopharmaceuticals.
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ELISA of milk whey from the Transgenic Goat BP21 and F1 Transgenic Goat.
2018Co-Authors: Ting Zhang, Lei Jiang, Minya Zhou, Yong ChengAbstract:ELISA of milk whey from the Transgenic Goat BP21 and F1 Transgenic Goat.
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FAPA analysis of partial whey from Transgenic Goat milk.
2018Co-Authors: Ting Zhang, Lei Jiang, Minya Zhou, Yong ChengAbstract:1–9: Reteplase standard (concentrations are 2,000, 1,500, 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μg/mL, respectively); 10–11: whey collected on different days from the Transgenic Goat BP21 (diluted 10-folds); 12: whey collected on different days from the F1 Transgenic Goat (diluted 10-folds); 13, 14: whey from non-Transgenic Goats as the negative control; 15: PBS as the negative control.
Qian Yang - One of the best experts on this subject based on the ideXlab platform.
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the effects of gh Transgenic Goats on the microflora of the intestine feces and surrounding soil
PLOS ONE, 2015Co-Authors: Qiang Zhang, Huiqing Yu, Jianquan Chen, Weiwei Hu, Qian Yang, Qinghua YuAbstract: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.
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Copy number and integration sites in growth hormone Transgenic Goats.
Genetics and molecular research : GMR, 2015Co-Authors: Qiang Zhang, Jian Lin, Qian YangAbstract:Transgenic Goats have been utilized for years to produce valuable protein. However, when Transgenic Goats are produced by random integration of inserted genes into cells, the copy number and integration sites of these genes in the Goat genome are typically indefinite. Most polymerase chain reaction (PCR)-based methods that have been utilized to determine copy number and integration sites of inserted genes in the genome require complicated manipulations. In this study, we used quantitative real-time PCR and thermal asymmetric interlaced-PCR to determine copy number and integration sites of the inserted genes, respectively. Copies of Transgenic Goat lines GHcd-2 and GHcd-7 were 12.95 ± 0.18 and 12.24 ± 1.12, respectively. Two integration sites, located in chromosomes 3 and 11 and referred to as tg1 and tg2, were identified by thermal asymmetric interlaced-PCR. Junction PCR was then performed to confirm the integration sites of growth hormone Transgenic Goats. Transgenic copy number and integration sites were determined, which will be useful for determining the relationship between the growth hormone expression, copy number, and integration sites.
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The copy number and integration site analysis of IGF-1 Transgenic Goat
International journal of molecular medicine, 2014Co-Authors: Jian Lin, Qiang Zhang, Li Q. Zhu, Qian YangAbstract:Transgenic animals have been used previously to study gene function, produce important proteins, and generate models for the study of human diseases. As the number of Transgenic species increases, reliable detection and molecular characterization of integration sites and copy number are crucial for confirming transgene expression and genetic stability, as well as for safety evaluation and to meet commercial demands. In this study, we generated four Transgenic Goats by somatic cell nuclear transfer (SCNT). After birth, the cloned Goat contained transferred insulin-like growth factor I (IGF-1) gene was initially confirmed using a polymerase chain reaction (PCR)-based method. The four cloned Goats were identified as IGF-1 Transgenic Goats by southern blotting. The number of copies of the IGF-1 gene in each of the Transgenic Goats was determined. Additionally, four integration sites of the transgene in the Transgenic Goats with a modified thermal asymmetric interlaced (TAIL)-PCR method were identified. The four different integration sites were located on chromosomes 2, 11, 16 and 18. The present study identified the copy number and integration sites using quantitative PCR (qPCR) and TAIL-PCR, enabling the bio- safety evaluation of the Transgenic Goats.
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Production GH Transgenic Goat improving mammogenesis by somatic cell nuclear transfer.
Molecular Biology Reports, 2014Co-Authors: J. Q. Chen, H. Q. Yu, X J Xu, Qinghua Yu, Qian YangAbstract: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.
Yin Zhen - One of the best experts on this subject based on the ideXlab platform.
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One-step purification of tissue-type plasminogen activator derived from expression by milk gland of Transgenic Goat using affinity chromatography.
Chinese journal of veterinary science, 2000Co-Authors: Huang Jian, Song Xinrong, Ren Qisheng, Yin ZhenAbstract:A tissue type plasminogen activator derived from expression by milk gland of Transgenic Goat was isolated and purificated. Precipitated with acetate acid and acetone in advance, the t PA in the milk was purificated using one step affinity chromatography with benzamidin sepharose 6B. The final product had a specific activity of 188 000 IU/mg protein. The overall activity recovery was about 26% with 6 714.3 fold increase in specific activity. Analysis by SDS PAGE electrophoresis in the presence of a reducing agent, the purity of t PA was over 95% with the two chain t PA to be occupied over 90% of it. Western blotting analysis showed that the product was possessed of the natural t PA antigen characteristics. Analysed with fibrin plate, the product had the same specific activity and molecular weight as natural t PA and could be inhibited by the t PA monoclonal antibody.
Qinghua Yu - One of the best experts on this subject based on the ideXlab platform.
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the effects of gh Transgenic Goats on the microflora of the intestine feces and surrounding soil
PLOS ONE, 2015Co-Authors: Qiang Zhang, Huiqing Yu, Jianquan Chen, Weiwei Hu, Qian Yang, Qinghua YuAbstract: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.
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Production GH Transgenic Goat improving mammogenesis by somatic cell nuclear transfer.
Molecular Biology Reports, 2014Co-Authors: J. Q. Chen, H. Q. Yu, X J Xu, Qinghua Yu, Qian YangAbstract: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.