-
Hongqin Zhuang,
Weiwei Jiang,
Xiangyu Zhang,
Fan Qiu,
Ziyi Gan,
Wei Cheng,
Jing Zhang,
Shengwen Guan, Bo Tang,
Qilai Huang,
Xinhua Wu,
Xiaofeng Huang,
Wenhui Jiang,
Qingang Hu,
Min Lu,
Zi-Chun Hua
[show abstract]
[hide abstract]
ABSTRACT: Many cancer cell types are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Here, we examined whether HSP70 suppression by small interfering RNA (siRNA) sensitized non-small cell lung cancer (NSCLC) cells to TRAIL-induced apoptosis and the underlying mechanisms. We demonstrated that HSP70 suppression by siRNA sensitized NSCLC cells to TRAIL-induced apoptosis by upregulating the expressions of death receptor 4 (DR4) and death receptor 5 (DR5) through activating NF-κB, JNK, and, subsequently, p53, consequently significantly amplifying TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Consistently, the pro-apoptotic proteins Bad and Bax were upregulated, while the anti-apoptotic protein Bcl-2 was downregulated. The luciferase activity of the DR4 promoter was blocked by a NF-κB pathway inhibitor BAY11-7082, suggesting that NF-κB activation plays an important role in the transcriptional upregulation of DR4. Additionally, HSP70 suppression inhibited the phosphorylation of ERK, AKT, and PKC, thereby downregulating c-FLIP-L. A549 xenografts in mice receiving HSP70 siRNA showed TRAIL-induced cell death and increased DR4/DR5 levels and reduced tumor growth. The combination of psiHSP70 gene therapy with TRAIL also significantly increased the survival benefits induced by TRAIL therapy alone. Interestingly, HSP27 siRNA and TRAIL together could not suppress tumor growth or prolong the survival of tumor-bearing mice significantly, although the combination could efficiently induce the apoptosis of A549 cells in vitro. Our findings suggest that HSP70 suppression or downregulation might be promising to overcome TRAIL resistance in cancer.
Journal of Molecular Medicine 09/2012; · 4.67 Impact Factor
-
Guochun Gong,
Yunping Dai,
Baoliang Fan,
Huabing Zhu,
Lili Wang,
Haiping Wang, Bo Tang,
Ying Liu,
Rong Li,
Rong Wan,
Yinghua Huang,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector (pCE-EGFP-IRES-Neo-dNdB)
containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was carried out
using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed
to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves, and 5 (29.4%) recipients
were found to be pregnant. Three of them maintained to term and delivered three cloned calves. PCR and Southern blot analysis
confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in
biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic
calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could
be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker
selective vector.
Chinese Science Bulletin 04/2012; 49(2):161-166. · 1.32 Impact Factor
-
Shen Liu,
Xiangqing Li,
Dan Lu,
Shengzhe Shang,
Meili Wang,
Min Zheng,
Ran Zhang, Bo Tang,
Qiuyan Li,
Yunping Dai,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: Transgenesis has been used for expressing human lysozyme (hLZ) in the milk of livestock to improve their disease resistance. Here we describe a human lactoferrin (hLF) BAC as a candidate vector for high-level expression of hLZ in the milk of transgenic mice. Using recombineering, hLF genomic DNA in the hLF BAC was replaced by the hLZ gene (from the ATG start codon to the TAA stop codon), and flanking regions of the hLF gene (a 90-kb 5' and a 30-kb 3') were used as transcriptional control elements for hLZ expression. When this construct was used to generate transgenic mice, rhLZ was highly expressed in the milk of four transgenic mouse lines (1.20-1.76 g/L), was expressed at a lower level in one additional line (0.21 g/L). rhLZ from the milk of these transgenic mice exhibited the same antibacterial activity as native hLZ. Our results suggest a potential approach for producing large amounts of hLZ in the milk of livestock.
Transgenic Research 07/2011; 21(2):407-14. · 2.75 Impact Factor
-
Bin Yang,
Jianwu Wang, Bo Tang,
Yufang Liu,
Chengdong Guo,
Penghua Yang,
Tian Yu,
Rong Li,
Jianmin Zhao,
Lei Zhang,
Yunping Dai,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: There is great potential for using transgenic technology to improve the quality of cow milk and to produce biopharmaceuticals within the mammary gland. Lysozyme, a bactericidal protein that protects human infants from microbial infections, is highly expressed in human milk but is found in only trace amounts in cow milk.
We have produced 17 healthy cloned cattle expressing recombinant human lysozyme using somatic cell nuclear transfer. In this study, we just focus on four transgenic cattle which were natural lactation. The expression level of the recombinant lysozyme was up to 25.96 mg/L, as measured by radioimmunoassay. Purified recombinant human lysozyme showed the same physicochemical properties, such as molecular mass and bacterial lysis, as its natural counterpart. Moreover, both recombinant and natural lysozyme had similar conditions for reactivity as well as for pH and temperature stability during in vitro simulations. The gross composition of transgenic and non-transgenic milk, including levels of lactose, total protein, total fat, and total solids were not found significant differences.
Thus, our study not only describes transgenic cattle whose milk offers the similar nutritional benefits as human milk but also reports techniques that could be further refined for production of active human lysozyme on a large scale.
PLoS ONE 01/2011; 6(3):e17593. · 4.09 Impact Factor
-
Yan Liu,
Qian Wu,
Huiting Cui,
Qinghe Li,
Yiqiang Zhao,
Juan Luo,
Qiuyue Liu,
Xiuzhu Sun, Bo Tang,
Lei Zhang,
Yunping Dai,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: Both enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase type II enzyme (NPTII) are widely used in transgenic studies, but their side effects have not been extensively investigated. In this study, we evaluated the expression profiles of the two marker genes and the relationship between their expression and organ abnormalities. Eight transgenic cloned cattle were studied, four harboring both EGFP and NPTII, and four harboring only the NPTII gene. Four age-matched cloned cattle were used as controls. EGFP and NPTII expression were measured and detected by Q-PCR, Western blot, ELISA, and RIA in heart, liver, and lungs, and the values ranged from 0.3 to 5 microg/g. The expression profiles exhibited differential or mosaic pattern between the organs, the pathologic symptoms of which were identified, but were similar to those of age-matched cloned cattle. All data indicated that the expression of EGFP and NPTII is not associated with organ abnormalities in transgenic cloned cattle.
Cloning and Stem Cells 10/2008; 10(4):421-8. · 2.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells.
Transgenic Research 09/2008; 17(4):727-32. · 2.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Catalase plays an important role in protecting organisms against oxidative damage caused by reactive oxygen species (ROS) by degrading surplus hydrogen peroxide. Addition of exogenous catalase can alleviate injuries caused by ROS. Thus, production of human catalase through genetic engineering will meet the increasing therapeutic demand for this enzyme. In this study, we successfully expressed the recombinant gene in mouse mammary gland, and biologically active human catalase was secreted into the milk of the transgenic mice. The peroxisomal targeting sequence (PTS) within the catalase gene had no significant negative effect on the secretion of the recombinant protein. Intake of the transgenic milk by the pups was found to decrease lipid peroxidation, increase the total superoxide dismutase (T-SOD) activity in the brain, and enhance the total antioxidative capacity (T-AOC) of brain, liver, and serum. To our knowledge, this is the first example of efficient production of biologically active human catalase in the milk of transgenic animals. Our study suggests that scaled-up production in transgenic farm animals would yield sufficient human catalase for biomedical research and clinical therapies.
Free Radical Biology and Medicine 08/2008; 45(8):1135-42. · 5.42 Impact Factor
-
Penghua Yang,
Jianwu Wang,
Guochun Gong,
Xiuzhu Sun,
Ran Zhang,
Zhuo Du,
Ying Liu,
Rong Li,
Fangrong Ding, Bo Tang,
Yunping Dai,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79 x 10(-2) percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.
PLoS ONE 02/2008; 3(10):e3453. · 4.09 Impact Factor
-
Shuyang Yu,
Mifang Liang,
Baoliang Fan,
Hongtao Xu,
Chuan Li,
Quanfu Zhang,
Dexin Li, Bo Tang,
Shijie Li,
Yunping Dai,
Meili Wang,
Min Zheng,
Bingxue Yan,
Qinghong Zhu,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: Transgenic mice expressing a recombinant human monoclonal antibody (rHMAb) against hantavirus were generated. These mice could be used as models to explore the possibilities of producing rHMAbs for therapeutic purposes. The highest concentration of the rHMAb in the milk of the transgenic females was 6.6 mg/ml. The rHMAb was also detected in the sera of pups fed by the transgenic females. Both the rHMAbs in the milk of transgenic mice and those in the sera of suckling pups were found to be active against hantaviruses, although the light chain of the antibody absorbed by the pups was modified by N-linked glycosylation.
Journal of Virology 05/2006; 80(8):4183-6. · 5.40 Impact Factor
-
Guochun Gong,
Yunping Dai,
Baoliang Fan,
Huabing Zhu,
Shien Zhu,
Haiping Wang,
Lili Wang, Bo Tang,
Rong Li,
Rong Wan,
Ying Liu,
Yinhua Huang,
Lei Zhang,
Xiuzhu Sun,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: The present study examined effects of genetic manipulation and serum starvation on in vitro developmental potential of bovine somatic cell nuclear transfer (SCNT) embryos and vitrification on in vivo developmental competence of transgenic SCNT blastocysts. Fetal oviduct epithelial cells (FOECs) were isolated from the oviduct of a Day 147 bovine fetus and transfected with a plasmid (pCE-EGFP-IRES-NEO) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes. There were no significant differences (P > 0.05) in cleavage rates or development rates to the blastocyst stage for SCNT embryos derived from FOECs (72.5 and 47.8%, respectively) or transfected FOECs (TFOECs, 73.8 and 47.7%, respectively); nor from serum-fed (73.6 and 47.2%, respectively) or serum-starved (72.7 and 48.3%, respectively) cells. Seventeen of Day 7 GFP-embryos (eight fresh blastocysts and nine vitrified/thawed blastocysts ) were transferred to recipients with one embryo per recipient. Two (25%) recipients were confirmed pregnant at Day 60 in fresh blastocysts group, and three recipients (33%) were confirmed pregnant at Day 60 in vitrified/thawed blastocysts group. Two healthy calves (25%) were obtained from fresh blastocysts and one (11%) from vitrified/thawed blastocysts. Microsatellite analysis confirmed that the three clones were genetically identical to the donor cells. Moreover, PCR and Southern blot demonstrated integration of transgene in genomic DNA of all three cloned calves. Expression of GFP in skin biopsies isolated from transgenic cloned calves and fibroblasts derived from the skin biopsies revealed the activity of EGFP gene, and G418 resistance in vitro of these fibroblasts confirmed the activity of Neor gene. Our results show that genetic manipulation and serum starvation of donor cells (FOECs) do not affect in vitro developmental competence of bovine SCNT embryos, and vitrified transgenic SCNT blastocysts can develop to term successfully.
Molecular Reproduction and Development 11/2004; 69(3):278-88. · 2.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Microsatellite markers are widely used in linkage mapping, parentage testing, population genetic studies, and molecular evolution studies in many agricultural species, while only a limited number of ostrich (Struthio camelus) microsatellites have been isolated. Thus, we constructed a random small-insert genomic library and a microsatellite-enriched library containing CA repeats. Fourteen clones containing CA repeats were isolated from 3462 clones in the non-enriched library by radioactive screening and 248 positive clones were isolated from 300 sequenced clones from the enriched library by PCR screening. After the enrichment procedures, the proportion of clones containing CA repeats was raised to 78.8%, compared with 0.4% in the non-enriched libraries, indicating that the enrichment value approaches 200 fold, which decreased the time and cost of cloning. The number of complete simple CA repeats in these positive clones ranged from 5 to 29. The primers for 94 of these microsatellites were developed and used to detect polymorphisms, of which 61 loci exhibited length polymorphisms in 17 unrelated ostrich individuals. The new polymorphic microsatellite markers we have identified and characterized will contribute to the ostrich genetic map, parentage testing, and comparative genomics between avian species.
Genome 11/2003; 46(5):833-40. · 1.65 Impact Factor
-
Yinhua Huang,
Jianfeng Tu,
Xuebo Cheng, Bo Tang,
Xiaoxiang Hu,
Zhaoliang Liu,
Jidong Feng,
Yankun Lou,
Li Lin,
Ke Xu,
Yulong Zhao,
Ning Li
[show abstract]
[hide abstract]
ABSTRACT: In order to study duck microsatellites, we constructed a library enriched for (CA)n, (CAG)n, (GCC)n and (TTTC)n. A total of 35 pairs of primers from these microsatellites were developed and used to detect polymorphisms in 31 unrelated Peking ducks. Twenty-eight loci were polymorphic and seven loci were monomorphic. A total of 117 alleles were observed from these polymorphic microsatellite markers, which ranged from 2 to 14 with an average of 4.18 per locus. The frequencies of the 117 alleles ranged from 0.02 to 0.98. The highest heterozygosity (0.97) was observed at the CAUD019 microsatellite locus and the lowest heterozygosity (0.04) at the CAUD008 locus, and 11 loci had heterozygosities greater than 0.50 (46.43%). The polymorphism information content (PIC) of 28 loci ranged from 0.04 to 0.88 with an average of 0.42. All the above markers were used to screen the polymorphism in other bird species. Two markers produced specific monomorphic products with the chicken DNA. Fourteen markers generated specific fragments with the goose DNA: 5 were polymorphic and 9 were monomorphic. But no specific product was detected with the peacock DNA. Based on sequence comparisons of the flanking sequence and repeat, we conclude that 2 chicken loci and 14 goose loci were true homologous loci of the duck loci. The microsatellite markers identified and characterized in the present study will contribute to the genetic map, quantitative traits mapping, and phylogenetic analysis in the duck and goose.
Genetics Selection Evolution 37(4):455-72. · 2.88 Impact Factor
