WorldWideScience

Sample records for human cd7 transgene

  1. Expression of human CD7 cDNA in human embryonic kidney 293 cells%人CD7 cDNA在HEK293细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    周艳春; 朱锡华; 黄云辉

    2001-01-01

    目的建立表达人白细胞分化抗原CD7蛋白的哺乳动物细胞模型,为深入研究CD7的功能奠定基础.方法采用PCR方法扩增CD7 cDNA,将目的片段定向克隆于真核表达载体pcDNA3,限制性内切酶酶切分析重组质粒并进行序列测定;通过电穿孔法将重组真核表达载体pcDNA3/CD7转染于人胚肾293细胞(HEK293),经G418筛选得到抗性克隆;利用FCM检测CD7分子在HEK293细胞的表达.结果得到了含CD7全长cDNA的重组真核表达载体pcDNA3/CD7.FCM分析表明:转染了pcDNA3/CD7的HEK293细胞表达人CD7蛋白.结论为深入研究CD7的作用机制提供了条件.

  2. Dissecting the Entry Route of Saporin-based a-CD7 Immunotoxins in Human T-Cell Acute Lymphoblastic Leukaemia Cells

    Directory of Open Access Journals (Sweden)

    Maria Serena Fabbrini

    2013-01-01

    Full Text Available Elucidating the intracellular fate(s of targeted toxins is of fundamental importance for their optimal use as anticancer drugs, since the biochemical targets of their enzymatic activity reside in the cell cytoplasm, as in the case of the plant ribosome inactivating proteins (RIP saporin, ricin and of bacterial toxins. In this paper, we compared the cell surface binding and cytotoxic properties of the model RIP ricin to an immunotoxin constructed with a monoclonal antibody directed against the human T-cell marker CD7 covalently linked to saporin (CD7-SAP. Our results indicate that, despite the fact that internalization takes place via an apparently common entry route leading to the Golgi complex, surprisingly, the addition of an endoplasmic reticulum retrieval C-terminal signal (KDEL to CD7-SAP does not potentiate its cytotoxicity. In addition, while ricin toxicity is clearly reduced by Brefeldin A under conditions where this fungal metabolite causes Golgi stack disruption, we paradoxically observed a potentiating effect by Brefeldin A on CD7-SAP cytotoxicity suggesting that this inhibitor interferes with retrograde route(s other than the well established Trans-Golgi Network-ER retrograde route.

  3. Transgenic rabbits as therapeutic protein bioreactors and human disease models.

    Science.gov (United States)

    Fan, Jianglin; Watanabe, Teruo

    2003-09-01

    Genetically modified laboratory animals provide a powerful approach for studying gene expression and regulation and allow one to directly examine structure-function and cause-and-effect relationships in pathophysiological processes. Today, transgenic mice are available as a research tool in almost every research institution. On the other hand, the development of a relatively large mammalian transgenic model, transgenic rabbits, has provided unprecedented opportunities for investigators to study the mechanisms of human diseases and has also provided an alternative way to produce therapeutic proteins to treat human diseases. Transgenic rabbits expressing human genes have been used as a model for cardiovascular disease, AIDS, and cancer research. The recombinant proteins can be produced from the milk of transgenic rabbits not only at lower cost but also on a relatively large scale. One of the most promising and attractive recombinant proteins derived from transgenic rabbit milk, human alpha-glucosidase, has been successfully used to treat the patients who are genetically deficient in this enzyme. Although the pronuclear microinjection is still the major and most popular method for the creation of transgenic rabbits, recent progress in gene targeting and animal cloning has opened new avenues that should make it possible to produce transgenic rabbits by somatic cell nuclear transfer in the future. Based on a computer-assisted search of the studies of transgenic rabbits published in the English literature here, we introduce to the reader the achievements made thus far with transgenic rabbits, with emphasis on the application of these rabbits as human disease models and live bioreactors for producing human therapeutic proteins and on the recent progress in cloned rabbits.

  4. Generation and characterization of human heme oxygenase-1 transgenic pigs.

    Directory of Open Access Journals (Sweden)

    Hye-Jung Yeom

    Full Text Available Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1, an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs. Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

  5. Generation and characterization of human heme oxygenase-1 transgenic pigs.

    Science.gov (United States)

    Yeom, Hye-Jung; Koo, Ok Jae; Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J; Kim, Hyunil; Surh, Charles D; Lee, Byeong-Chun; Ahn, Curie

    2012-01-01

    Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

  6. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    OpenAIRE

    Meng-Hwan Lee; Yin-Shen Lin; Ching-Fu Tu; Chon-Ho Yen

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitat...

  7. [Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

    Science.gov (United States)

    Li, Lan; Shen, Wei; Pan, Qing-Yu; Min, Ling-Jiang; Sun, Yu-Jiang; Fang, Yong-Wei; Deng, Ji-Xian; Pan, Qing-Jie

    2006-12-01

    Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

  8. Recombinant human factor IX produced from transgenic porcine milk.

    Science.gov (United States)

    Lee, Meng-Hwan; Lin, Yin-Shen; Tu, Ching-Fu; Yen, Chon-Ho

    2014-01-01

    Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  9. Recombinant Human Factor IX Produced from Transgenic Porcine Milk

    Directory of Open Access Journals (Sweden)

    Meng-Hwan Lee

    2014-01-01

    Full Text Available Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa. The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX. The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

  10. Transgenic rabbit that expresses a functional human lipoprotein (a)

    Science.gov (United States)

    Rouy, Didier; Duverger, Nicolas; Emmanuel, Florence; Denefle, Patrice; Houdebine, Louis-Marie; Viglietta, Celine; Rubin, Edward M.; Hughes, Steven D.

    2003-01-01

    A transgenic rabbit which has in its genomic DNA sequences that encode apolipoprotein (a) and apolipoprotein B polypeptides which are capable of combining to produce lipoprotein (a), a process for creating such a rabbit, and the use of the rabbit to identify compounds which are effective in the treatment of human diseases which are associated with, induced and/or exacerbated by Lp(a) expression.

  11. Human mutant huntingtin disrupts vocal learning in transgenic songbirds.

    Science.gov (United States)

    Liu, Wan-Chun; Kohn, Jessica; Szwed, Sarah K; Pariser, Eben; Sepe, Sharon; Haripal, Bhagwattie; Oshimori, Naoki; Marsala, Martin; Miyanohara, Atsushi; Lee, Ramee

    2015-11-01

    Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical-basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

  12. [Generation of transgenic mice expressing human lysozyme in mammary gland].

    Science.gov (United States)

    Yan, Hua; Li, Guo-cai; Sun, Huai-chang

    2005-10-01

    To evaluate the feasibility of generating animal mammary gland bioreactors expressing human lysozyme (hLYZ). The recombinant vector p205C3-hLYZ, as a result of connecting the hLYZ cDNA with the mammry gland expression vector p205C3, was used to generate transfer genic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 females and 5 males) and 4 (1 females and 3 males) were found to contain the transfer-gene by PCR and Southern blotting respectively. The results of Western blotting indicated that the expressed protein had the same molecular weight as that of normal hLYZ. From the F1 generation on, the mice mated only with their brothers or sisters and a colony of F7 transgenic mice was obtained. Among the offspring, the female transgenic mice maintained and expressed the transfer-gene stably with an expression level as high as 750 mg/L. The expressed protein had strong tissue specificity, and in addition to the mammary glands, some degree of ectropic expression in the spleens and intestines of the transgenic mice was confirmed by dot blotting assay. These data indicate that the mice mammary gland bioreactors expressing hLYZ have been successfully generated.

  13. Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits

    OpenAIRE

    2010-01-01

    While there are many reports in the literature describing the attributes of specific applications of transgenic animals for agriculture, there are relatively few studies focusing on the fitness of the transgenic animals themselves. This work was designed to gather information on genetically modified food animals to determine if the presence of a transgene can impact general animal production traits. More specifically, we used a line of transgenic dairy goats expressing human lysozyme in their...

  14. Evaluating the fitness of human lysozyme transgenic dairy goats: growth and reproductive traits.

    Science.gov (United States)

    Jackson, Kathryn A; Berg, Jolene M; Murray, James D; Maga, Elizabeth A

    2010-12-01

    While there are many reports in the literature describing the attributes of specific applications of transgenic animals for agriculture, there are relatively few studies focusing on the fitness of the transgenic animals themselves. This work was designed to gather information on genetically modified food animals to determine if the presence of a transgene can impact general animal production traits. More specifically, we used a line of transgenic dairy goats expressing human lysozyme in their mammary gland to evaluate the reproductive fitness and growth and development of these animals compared to their non-transgenic counterparts and the impact of consuming a transgenic food product, lysozyme-containing milk. In males, none of the parameters of semen quality, including semen volume and concentration, total sperm per ejaculate, sperm morphology, viability and motility, were significantly different between transgenic bucks and non-transgenic full-sib controls. Likewise, transgenic females of this line did not significantly differ in the reproductive traits of gestation length and litter size compared to their non-transgenic counterparts. To evaluate growth, transgenic and non-transgenic kid goats received colostrum and milk from either transgenic or non-transgenic does from birth until weaning. Neither the presence of the transgene nor the consumption of milk from transgenic animals significantly affected birth weight, weaning weight, overall gain and post-wean gain. These results indicate that the analyzed reproductive and growth traits were not regularly or substantially impacted by the presence or expression of the transgene. The evaluation of these general parameters is an important aspect of defining the safety of applying transgenic technology to animal agriculture.

  15. Expression of recombinant human lysozyme in the milk of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    YU Zhengquan; FAN Baoliang; DAI Yunping; ZHENG Ming; NIU Huiling; WANG Meili; WANG Lili; FEI Jing; LI Ning

    2003-01-01

    Human lysozyme is a 130-aa (amino acid) alkaline polypeptide, and has both anti-bacterial and anti-viral properties which make it an important component of human natural immunity system. As a first step toward the ultimate goal ofimproving the anti-bacterial properties of bovine and ovine milk, a transgenic mouse that contains the genomic DNA sequence of the human lysozme gene has been generated for the first time. From 83 mice generated by microinjection, a total of 6 positive transgenic mice were identified by PCR and Southern blot. F1 mice positive for transgene in lines were also detected by PCR. This shows that transgene could be transmitted from founder transgenic mice to their offspring. Recombinant human lysozyme (rHlys) was found in the whey of 3 female positive transgenic mice by Western blot. The highest concentration of rHlys for transgenic micewas 0.2 mg/mL. The antibacterial activity of the whey for transgenic mice was highly enhanced up to 0.4 times as much as that of human, while that of non-transgenic mouse was very low. Although the lysozyme activity of transgenic mice is still lower than that of human, the rHlys exhibits the same specific activity as that of human lysozyme. It provides a strong basis for further studies into the possible application of rHlys express in mammary gland.

  16. Introducing Human APOE into Aβ Transgenic Mouse Models

    Directory of Open Access Journals (Sweden)

    Leon M. Tai

    2011-01-01

    Full Text Available Apolipoprotein E (apoE and apoE/amyloid-β (Aβ transgenic (Tg mouse models are critical to understanding apoE-isoform effects on Alzheimer's disease risk. Compared to wild type, apoE−/− mice exhibit neuronal deficits, similar to apoE4-Tg compared to apoE3-Tg mice, providing a model for Aβ-independent apoE effects on neurodegeneration. To determine the effects of apoE on Aβ-induced neuropathology, apoE−/− mice were crossed with Aβ-Tg mice, resulting in a significant delay in plaque deposition. Surprisingly, crossing human-apoE-Tg mice with apoE−/−/Aβ-Tg mice further delayed plaque deposition, which eventually developed in apoE4/Aβ-Tg mice prior to apoE3/Aβ-Tg. One approach to address hAPOE-induced temporal delay in Aβ pathology is an additional insult, like head injury. Another is crossing human-apoE-Tg mice with Aβ-Tg mice that have rapid-onset Aβ pathology. For example, because 5xFAD mice develop plaques by 2 months, the prediction is that human-apoE/5xFAD-Tg mice develop plaques around 6 months and 12 months before other human-apoE/Aβ-Tg mice. Thus, tractable models for human-apoE/Aβ-Tg mice continue to evolve.

  17. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O;

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific tra...... of the transgene was observed in cell types other than beta-islet cells.......Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...

  18. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    Science.gov (United States)

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF.

    Directory of Open Access Journals (Sweden)

    Yonghua He

    Full Text Available Necrotizing enterocolitis (NEC is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF, typically found in bodily fluids, such as amniotic fluid, salvia and mother's breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N' terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.

  20. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies.

    Science.gov (United States)

    Gomes-Silva, Diogo; Srinivasan, Madhuwanti; Sharma, Sandhya; Lee, Ciaran M; Wagner, Dimitrios L; Davis, Timothy H; Rouce, Rayne H; Bao, Gang; Brenner, Malcolm K; Mamonkin, Maksim

    2017-07-20

    Extending the success of chimeric antigen receptor (CAR) T cells to T-cell malignancies is problematic because most target antigens are shared between normal and malignant cells, leading to CAR T-cell fratricide. CD7 is a transmembrane protein highly expressed in acute T-cell leukemia (T-ALL) and in a subset of peripheral T-cell lymphomas. Normal expression of CD7 is largely confined to T cells and natural killer (NK) cells, reducing the risk of off-target-organ toxicity. Here, we show that the expression of a CD7-specific CAR impaired expansion of transduced T cells because of residual CD7 expression and the ensuing fratricide. We demonstrate that targeted genomic disruption of the CD7 gene prevented this fratricide and enabled expansion of CD7 CAR T cells without compromising their cytotoxic function. CD7 CAR T cells produced robust cytotoxicity against malignant T-cell lines and primary tumors and were protective in a mouse xenograft model of T-ALL. Although CD7 CAR T cells were also toxic against unedited (CD7(+)) T and NK lymphocytes, we show that the CD7-edited T cells themselves can respond to viral peptides and therefore could be protective against pathogens. Hence, genomic disruption of a target antigen overcomes fratricide of CAR T cells and establishes the feasibility of using CD7 CAR T cells for the targeted therapy of T-cell malignancies. © 2017 by The American Society of Hematology.

  1. The human apoE7 and apoE4 transgenic mice models

    Institute of Scientific and Technical Information of China (English)

    孙明增; 琦祖和

    2001-01-01

    To scrutinize the disorders caused by human mutant apoE7/apoE4, human apoE4 and E7 transgenic mice were established with microinjection technique to examine molecular genetic phenomena in vivo. The integration and expression of h-apoE mutant genes in transgenic mice were determined with Southern blot, Northern blot and ELISA. The current studies indicated that the transgenes and the phenotypes regarding expression of transgenes could be transmitted stably in transgenic lines. The levels of serum lipid in transgenic mice showed the characteristics of hyperlipidemia. Besides, behavior tests demonstrated the degeneration of learning and memory in transgenic mice. Short life span was observed in 2 transgenic lines. After fed with high lipid food high serum lipid was found both in normal and transgenic mice, but their mechanism regulating lipid metabolism was different. It was also verified that the human apoE mutants located at either N-terminal or C-terminal had the same pathogenesis regarding disorders of lipid metabolism in murine.

  2. Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Kwang Sung; Won, Ji Young [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Park, Jin-Ki [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Sorrell, Alice M. [Department of Physiology, Dankook University School of Medicine, Cheonan (Korea, Republic of); Heo, Soon Young; Kang, Jee Hyun [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Woo, Jae-Seok [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Choi, Bong-Hwan [Genomics and Bioinformatics Division, National Institute of Animal Science, Suwon (Korea, Republic of); Chang, Won-Kyong [Animal Biotechnology Division, National Institute of Animal Science, Suwon (Korea, Republic of); Shim, Hosup, E-mail: shim@dku.edu [Department of Nanobiomedical Science, Dankook University, Cheonan (Korea, Republic of); Institute of Tissue Regeneration Engineering, Dankook University, Cheonan (Korea, Republic of)

    2010-10-01

    Research highlights: {yields} Human CD59 (hCD59) gene was introduced into porcine embryonic germ (EG) cells. {yields} hCD59-transgenic EG cells were resistant to hyperacute rejection in cytolytic assay. {yields} hCD59-transgenic pigs were produced by EG cell nuclear transfer. -- Abstract: This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.

  3. Expression of human apolipoprotein B and assembly of lipoprotein(a) in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Callow, M.J.; Stoltzfus, L.J.; Rubin, E.M. [Lawrence Berkeley Lab., CA (United States); Lawn, R.M. [Stanford Univ., CA (United States)

    1994-03-15

    The atherogenic macromolecule lipoprotein(a) [Lp(a)] has resisted in vivo analyses partly because it is found in a limited number of experimental animals. Although transgenic mice expressing human apolipoprotein (a) [apo(a)] have previously been described, they failed to assemble Lp(a) particles because of the inability of human apo(a) to associate with mouse apolipoprotein B (apoB). The authors isolated a 90-kilobase P1 phagemid containing the human apoB gene and with this DNA generated 13 lines of transgenic mice of which 11 expressed human apoB. The human apoB transcript was expressed and edited in the liver of the transgenic mice. Plasma concentrations of human apoB, as well as low density lipoprotein (LDL), were related to transgene copy number; the transgenic line with the most copies of human apoB had a >4-fold increase in LDL cholesterol compared with nontransgenics and a lipoprotein profile similar to that of humans. When human apoB and apo(a) transgenic mice were bred together, plasma apo(a) in mice expressing both human proteins was tightly associated with lipoproteins in the LDL density region. These studies demonstrate the successful expression of human apoB and the efficient assembly of Lp(a) in mice.

  4. Transgenic chickens expressing human urokinase-type plasminogen activator.

    Science.gov (United States)

    Lee, Sung Ho; Gupta, Mukesh Kumar; Ho, Young Tae; Kim, Teoan; Lee, Hoon Taek

    2013-09-01

    Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

  5. Transgenic Nonhuman Primate Models for Human Diseases: Approaches and Contributing Factors

    Institute of Scientific and Technical Information of China (English)

    Yongchang Chen; Yuyu Niu; Weizhi Ji

    2012-01-01

    Nonhuman primates (NHPs) provide powerful experimental models to study human development,cognitive functions and disturbances as well as complex behavior,because of their genetic and physiological similarities to humans.Therefore,NHPs are appropriate models for the study of human diseases,such as neurodegenerative diseases including Parkinson's,Alzheimer's and Huntington's diseases,which occur as a result of genetic mutations.However,such diseass afflicting humans do not occur naturally in NHPs.So transgenic NHPs need to be established to understand the etiology of disease pathology and pathogenesis.Compared to rodent genetic models,the generation of transgenic NHPs for human diseases is inefficient,and only a transgenic monkey model for Huntington's disease has been reported.This review focuses on potential approaches and contributing factors for generating transgenic NHPs to study human diseases.

  6. Aberrant phenotypes of transgenic mice expressing dimeric human erythropoietin

    Directory of Open Access Journals (Sweden)

    Yun Seong-Jo

    2012-01-01

    Full Text Available Abstract Background Dimeric human erythropoietin (dHuEPO peptides are reported to exhibit significantly higher biological activity than the monomeric form of recombinant EPO. The objective of this study was to produce transgenic (tg mice expressing dHuEPO and to investigate the characteristics of these mice. Methods A dHuEPO-expressing vector under the control of the goat beta-casein promoter, which produced a dimer of human EPO molecules linked by a 2-amino acid peptide linker (Asp-Ile, was constructed and injected into 1-cell fertilized embryos by microinjection. Mice were screened using genomic DNA samples obtained from tail biopsies. Blood samples were obtained by heart puncture using heparinized tubes, and hematologic parameters were assessed. Using the microarray analysis tool, we analyzed differences in gene expression in the spleens of tg and control mice. Results A high rate of spontaneous abortion or death of the offspring was observed in the recipients of dHuEPO embryos. We obtained 3 founder lines (#4, #11, and #47 of tg mice expressing the dHuEPO gene. However, only one founder line showed stable germline integration and transmission, subsequently establishing the only transgenic line (#11. We obtained 2 F1 mice and 3 F2 mice from line #11. The dHuEPO protein could not be obtained because of repeated spontaneous abortions in the tg mice. Tg mice exhibited symptoms such as short lifespan and abnormal blood composition. The red blood cell count, white blood cell count, and hematocrit levels in the tg mice were remarkably higher than those in the control mice. The spleens of the tg mice (F1 and F2 females were 11- and -21-fold larger than those of the control mice. Microarray analysis revealed 2,672 spleen-derived candidate genes; more genes were downregulated than upregulated (849/764. Reverse transcriptase-polymerase chain reaction (RT-PCR and quantitative real-time PCR (qRT-PCR were used for validating the results of the microarray

  7. Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice

    DEFF Research Database (Denmark)

    Fromont-Racine, M; Bucchini, D; Madsen, O

    1990-01-01

    Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific......, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50...... of the transgene was observed in cell types other than beta-islet cells....

  8. Novel CD7-specific nanobody-based immunotoxins potently enhanced apoptosis of CD7-positive malignant cells

    Science.gov (United States)

    Tang, Jinle; Li, Jialu; Zhu, Xuejun; Yu, Yuan; Chen, Dan; Yuan, Lei; Gu, Zhenyang; Zhang, Xingding; Qi, Lin; Gong, Zhishu; Jiang, Pengjun; Yu, Juhua; Meng, Huimin; An, Gangli

    2016-01-01

    Various CD7-targeting immunotoxins have been tested for its potential in treating CD7+ malignant patients but none of those immunotoxins was approved clinically because of lacking enough efficacy and safety. Here we successfully constructed the monovalent and bivalent CD7 nanobody-based immunotoxins PG001 and PG002, both conjugated with a truncated derivative of Pseudomonas exotoxin A respectively. The prokaryotic system expressed immunotoxins not only maintained their binding specificity for CD7-positive cells with a Kd of 16.74 nM and 3.6 nM for PG001 and PG002 respectively, but also efficiently promoted antigen-restricted apoptosis of the CD7-positive leukemia cell lines Jurkat and CEM, and primary T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells with an in vitro cytotoxic activity (EC50) in the range of 23-30 pM for PG002. In NOD/SCID mice transplanted with CEM cells, PG001 and PG002 prevented engraftment of the cells and markedly prolonged mouse survival. Owing to the efficient antigen-restricted anti-leukemic activity of PG002, this CD7 nanobody-based immunotoxin exhibited a superior anti-CD7 positive malignancies activity than previously reported immunotoxins, and may represent a promising therapeutic strategy in treating CD7-positive leukemia and lymphoma, which still remain a significant clinical challenge. PMID:27083001

  9. Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide-sperm-mediated gene transfer.

    Science.gov (United States)

    Li, Lan; Shen, Wei; Min, Lingjiang; Dong, Huansheng; Sun, Yujiang; Pan, Qingjie

    2006-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins. However, it is difficult to validate whether these transgenic domestic animals are able to express the recombinant protein efficiently in their mammary glands before the birth of transgenic offspring. In the present study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed, and the expression of human lactoferrin (LF) gene was controlled by the goat beta-casein gene 5' flanking sequence. To obtain the most efficient transfection, the influence of DNA concentration, dimethylsulfoxide (DMSO) concentration, and the ratio of linear-to-circular DNA required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into rabbit spermatozoa was found to be efficient using 30 microg mL(-1) DNA, DMSO at a final concentration of 3%, and a 3 : 1 ratio of linear-to-circular DNA, with 29 of 85 (34.1%) in vitro-fertilised embryos being transgenic. Using DMSO-sperm-mediated gene transfer (DMSO-SMGT), 89 rabbit offspring were produced, with 46 of these (57.1%) being transgenic. As mammary gland bioreactor models, 17 of 21 (81%) transgenic female rabbits could express human LF protein in their glands. During lactation of the transgenic rabbits, the highest level of human LF protein expressed was 153 +/- 31 microg mL(-1), and the mean expression level in all of the transgenic rabbits was 103 +/- 20 microg mL(-1) in the third week, declining gradually after this time. Our results demonstrate that transgenic rabbits produced by DMSO-SMGT were able to express human LF protein in the correct tissue.

  10. Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

    Science.gov (United States)

    Hasegawa, Yoshinori; Ishikura, Tomoyuki; Hasegawa, Takanori; Watanabe, Takashi; Suzuki, Junpei; Nakayama, Manabu; Okamura, Yoshiaki; Okazaki, Tuneko; Koseki, Haruhiko; Ohara, Osamu; Ikeno, Masashi; Masumoto, Hiroshi

    2015-03-01

    The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

  11. The Impact of Transgenic Mosquitoes on Dengue Virulence to Humans and Mosquitoes

    OpenAIRE

    Medlock, Jan; Luz, Paula M; Struchiner,Claudio J.; Galvani, Alison P.

    2009-01-01

    Dengue is a major public health concern in the tropics and subtropics. Innovative transgenic strategies to render Aedes aegypti mosquitoes, the primary vector of dengue, incompetent for dengue transmission are under development. We modeled the evolutionary impact of different transgenic mosquito strategies on dengue-induced mortality, that is, dengue virulence, to both humans and mosquitoes. This model incorporates various evolutionary trade-offs in dengue virus epidemiological traits, for ex...

  12. CREATE TRANSGENIC RABBITS BY MICROINJECTING HUMAN apoA-ⅡGENE INTO FERTILIZED EGGS

    Institute of Scientific and Technical Information of China (English)

    Liu Enqi(刘恩岐); Shuji Kitajima; Masatoshi Morimoto

    2004-01-01

    Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods Superovulation and synchronization of estrus were induced in female Japanese White Rabbits by injecting hormone, then mating with male. After collected the fertilized eggs, the human apoA-Ⅱ gene was microinjected into the male pronucleus of eggs. The injected eggs were transferred into recipient female rabbits. Last, extract DNA from the new borns ear and determine whether the newborns were transgenic by polymerase chain reaction (PCR) or Southern blot analysis. Results A total of 822 embryos with microinjection of human apoAⅡ gene were implanted into 28 recipient rabbits. The number of surviving newborns was 37. 3 transgenic positive surviving founders were found with human apoA-Ⅱ.

  13. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene

    National Research Council Canada - National Science Library

    Wharram, Bryan L; Goyal, Meera; Wiggins, Jocelyn E; Sanden, Silja K; Hussain, Sabiha; Filipiak, Wanda E; Saunders, Thomas L; Dysko, Robert C; Kohno, Kenji; Holzman, Lawrence B; Wiggins, Roger C

    2005-01-01

    .... For determining the causal relationship between podocyte depletion and glomerulosclerosis, a transgenic rat strain in which the human diphtheria toxin receptor is specifically expressed in podocytes was developed...

  14. Transgenic bioreactors.

    Science.gov (United States)

    Jänne, J; Alhonen, L; Hyttinen, J M; Peura, T; Tolvanen, M; Korhonen, V P

    1998-01-01

    Since the generation of the first transgenic mice in 1980, transgene technology has also been successfully applied to large farm animals. Although this technology can be employed to improve certain production traits of livestock, this approach has not been very successful so far owing to unwanted effects encountered in the production animals. However, by using tissue-specific targeting of the transgene expression, it is possible to produce heterologous proteins in the extracellular space of large transgenic farm animals. Even though some recombinant proteins, such as human hemoglobin, have been produced in the blood of transgenic pigs, in the majority of the cases mammary gland targeted expression of the transgene has been employed. Using production genes driven by regulatory sequences of milk protein genes a number of valuable therapeutic proteins have been produced in the milk of transgenic bioreactors, ranging from rabbits to dairy cattle. Unlike bacterial fermentors, the mammary gland of transgenic bioreactors appear to carry out proper postsynthetic modifications of human proteins required for full biological activity. In comparison with mammalian cell bioreactors, transgenic livestock with mammary gland targeted expression seems to be able to produce valuable human therapeutic proteins at very low cost. Although not one transgenically produced therapeutic protein is yet on the market, the first such proteins have recently entered or even completed clinical trials required for their approval.

  15. Establishment of transgenic mice carrying gene encoding human zinc finger protein 191

    Science.gov (United States)

    Li, Jian-Zhong; Chen, Xia; Yang, Hua; Wang, Shui-Liang; Gong, Xue-Lian; Feng, Hao; Guo, Bao-Yu; Yu, Long; Wang, Zhu-Gang; Fu, Ji-Liang

    2004-01-01

    AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Krüppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo. PMID:14716836

  16. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    Science.gov (United States)

    Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  17. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    Directory of Open Access Journals (Sweden)

    Hanyu Wu

    Full Text Available Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies.

  18. Transgenic mice expressing human glucocerebrosidase variants: utility for the study of Gaucher disease.

    Science.gov (United States)

    Sanders, Angela; Hemmelgarn, Harmony; Melrose, Heather L; Hein, Leanne; Fuller, Maria; Clarke, Lorne A

    2013-08-01

    Gaucher disease is an autosomal recessively inherited storage disorder caused by deficiency of the lysosomal hydrolase, acid β-glucosidase. The disease manifestations seen in Gaucher patients are highly heterogeneous as is the responsiveness to therapy. The elucidation of the precise factors responsible for this heterogeneity has been challenging as the development of clinically relevant animal models of Gaucher disease has been problematic. Although numerous murine models for Gaucher disease have been described each has limitations in their specific utility. We describe here, transgenic murine models of Gaucher disease that will be particularly useful for the study of pharmacological chaperones. We have produced stable transgenic mouse strains that individually express wild type, N370S and L444P containing human acid β-glucosidase and show that each of these transgenic lines rescues the lethal phenotype characteristic of acid β-glucosidase null mice. Both the N370S and L444P transgenic models show early and progressive elevations of tissue sphingolipids with L444P mice developing progressive splenic Gaucher cell infiltration. We demonstrate the potential utility of these new transgenic models for the study of Gaucher disease pathogenesis. In addition, since these mice produce only human enzyme, they are particularly relevant for the study of pharmacological chaperones that are specifically targeted to human acid β-glucosidase and the common mutations underlying Gaucher disease.

  19. Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Wei SHEN; Lingjiang MIN; Qingyu PAN; Yujiang SUN; Jixian DENG; Qingjie PAN

    2008-01-01

    Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.

  20. Production of transgenic dairy goat expressing human α-lactalbumin by somatic cell nuclear transfer.

    Science.gov (United States)

    Feng, Xiujing; Cao, Shaoxian; Wang, Huili; Meng, Chunhua; Li, Jingxin; Jiang, Jin; Qian, Yong; Su, Lei; He, Qiang; Zhang, Qingxiao

    2015-02-01

    Production of human α-lactalbumin (hα-LA) transgenic cloned dairy goats has great potential in improving the nutritional value and perhaps increasing the yield of dairy goat milk. Here, a mammary-specific expression vector 5A, harboring goat β-lactoglobulin (βLG) promoter, the hα-LA gene, neo(r) and EGFP dual markers, was constructed. Then, it was effectively transfected into goat mammary epithelial cells (GMECs) and the expression of hα-LA was investigated. Both the hα-LA transcript and protein were detected in the transfected GMECs after the induction of hormonal signals. In addition, the 5A vector was introduced into dairy goat fetal fibroblasts (transfection efficiency ≈60-70%) to prepare competent transgenic donor cells. A total of 121 transgenic fibroblast clones were isolated by 96-well cell culture plates and screened with nested-PCR amplification and EGFP fluorescence. After being frozen for 8 months, the transgenic cells still showed high viabilities, verifying their ability as donor cells. Dairy goat cloned embryos were produced from these hα-LA transgenic donor cells by somatic cell nuclear transfer (SCNT), and the rates of fusion, cleavage, and the development to blastocyst stages were 81.8, 84.4, and 20.0%, respectively. A total of 726 reconstructed embryos derived from the transgenic cells were transferred to 74 recipients and pregnancy was confirmed at 90 days in 12 goats. Of six female kids born, two carried hα-LA and the hα-LA protein was detected in their milk. This study provides an effective system to prepare SCNT donor cells and transgenic animals for human recombinant proteins.

  1. Herbicide resistance of transgenic rice plants expressing human CYP1A1.

    Science.gov (United States)

    Kawahigashi, Hiroyuki; Hirose, Sakiko; Ohkawa, Hideo; Ohkawa, Yasunobu

    2007-01-01

    Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.

  2. Transgenic knockout mice with exclusively human sickle hemoglobinand sickle cell disease

    Energy Technology Data Exchange (ETDEWEB)

    Paszty, C.; Brion, C.; Manci, E.; Witkowska, E.; Stevens, M.; Narla, M.; Rubin, E.

    1997-06-13

    To create mice expressing exclusively human sicklehemoglobin (HbS), transgenic mice expressing human alpha-, gamma-, andbeta[S]-globin were generated and bred with knockout mice that haddeletions of the murine alpha- and beta-globin genes. These sickle cellmice have the major features (irreversibly sickled red cells, anemia,multiorgan pathology) found in humans with sickle cell disease and, assuch, represent a useful in vivo system to accelerate the development ofimproved therapies for this common genetic disease.

  3. Establishment of transgenic mice carrying the gene of human nuclear receptor NRSA2 (hB1F)

    Institute of Scientific and Technical Information of China (English)

    Shui-Liang Wang; Hua Yang; You-Hua Xie; Yuan Wang; Jian-Zhong Li; Long Wang; Zhu-Gang Wang; Ji-Liang Fu

    2003-01-01

    AIM: Human hepatitis B virus enhancer Ⅱ B1 binding factor (hB1F) was cloned and characterized as a novel member of the Ftz-F1 (NRSA) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hB1F transgenic mouse model to promote the functional study of hB1F. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice.The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2mice were identified by PCR analysis. RESULTS: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages.Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably. CONCLUSION: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

  4. Characterization of human antiviral adaptive immune responses during hepatotropic virus infection in HLA-transgenic human immune system mice.

    Science.gov (United States)

    Billerbeck, Eva; Horwitz, Joshua A; Labitt, Rachael N; Donovan, Bridget M; Vega, Kevin; Budell, William C; Koo, Gloria C; Rice, Charles M; Ploss, Alexander

    2013-08-15

    Humanized mice have emerged as a promising model to study human immunity in vivo. Although they are susceptible to many pathogens exhibiting an almost exclusive human tropism, human immune responses to infection remain functionally impaired. It has recently been demonstrated that the expression of HLA molecules improves human immunity to lymphotropic virus infections in humanized mice. However, little is known about the extent of functional human immune responses in nonlymphoid tissues, such as in the liver, and the role of HLA expression in this context. Therefore, we analyzed human antiviral immunity in humanized mice during a hepatotropic adenovirus infection. We compared immune responses of conventional humanized NOD SCID IL-2Rγ-deficient (NSG) mice to those of a novel NOD SCID IL-2Rγ-deficient strain transgenic for both HLA-A*0201 and a chimeric HLA-DR*0101 molecule. Using a firefly luciferase-expressing adenovirus and in vivo bioluminescence imaging, we demonstrate a human T cell-dependent partial clearance of adenovirus-infected cells from the liver of HLA-transgenic humanized mice. This correlated with liver infiltration and activation of T cells, as well as the detection of Ag-specific humoral and cellular immune responses. When infected with a hepatitis C virus NS3-expressing adenovirus, HLA-transgenic humanized mice mounted an HLA-A*0201-restricted hepatitis C virus NS3-specific CD8(+) T cell response. In conclusion, our study provides evidence for the generation of partial functional antiviral immune responses against a hepatotropic pathogen in humanized HLA-transgenic mice. The adenovirus reporter system used in our study may serve as simple in vivo method to evaluate future strategies for improving human intrahepatic immune responses in humanized mice.

  5. Effects of fenofibrate on hyperlipidemia and postprandial triglyceride metabolism in human apolipoprotein C1 transgenic mice

    NARCIS (Netherlands)

    Jong, M.C.; Dahlmans, V.E.H.; Princen, H.M.G.; Hofker, M.H.; Havekes, L.M.

    1998-01-01

    To study the in vivo role of apolipoprotein (apo) C1 in lipoprotein metabolism, we have generated transgenic mice expressing the human apo C1 gene. Apo C1 is a small 6.6 kDa protein that is primarily synthesized by the liver and is present on chylomicrons, very low density lipoproteins (VLDL) and hi

  6. Effects of fenofibrate on hyperlipidemia and postprandial triglyceride metabolism in human apolipoprotein C1 transgenic mice

    NARCIS (Netherlands)

    Jong, M.C.; Dahlmans, V.E.H.; Princen, H.M.G.; Hofker, M.H.; Havekes, L.M.

    1998-01-01

    To study the in vivo role of apolipoprotein (apo) C1 in lipoprotein metabolism, we have generated transgenic mice expressing the human apo C1 gene. Apo C1 is a small 6.6 kDa protein that is primarily synthesized by the liver and is present on chylomicrons, very low density lipoproteins (VLDL) and

  7. Adenohypophysial changes in mice transgenic for human growth hormone-releasing factor

    DEFF Research Database (Denmark)

    Stefaneanu, L; Kovacs, K; Horvath, E

    1989-01-01

    The effect of protracted GH-releasing factor (GRF) stimulation on adenohypophysial morphology was investigated in six mice transgenic for human GRF (hGRF). All animals had significantly higher plasma levels of GH and GRF and greater body weights than controls. Eight-month-old mice were killed...

  8. Developmental regulation of a complete 70kb human β-globin locus in transgenic mice.

    NARCIS (Netherlands)

    J. Strouboulis (John); N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1992-01-01

    textabstractWe have used a linker-based ligation strategy to combine two 35-kb cosmid inserts from the human beta-globin locus into one linear fragment containing the entire locus. This 70-kb fragment was introduced into transgenic mice by microinjection of fertilized eggs. Southern blot analysis sh

  9. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    Science.gov (United States)

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  10. The impact of transgenic mosquitoes on dengue virulence to humans and mosquitoes.

    Science.gov (United States)

    Medlock, Jan; Luz, Paula M; Struchiner, Claudio J; Galvani, Alison P

    2009-10-01

    Dengue is a major public health concern in the tropics and subtropics. Innovative transgenic strategies to render Aedes aegypti mosquitoes, the primary vector of dengue, incompetent for dengue transmission are under development. We modeled the evolutionary impact of different transgenic mosquito strategies on dengue-induced mortality, that is, dengue virulence, to both humans and mosquitoes. This model incorporates various evolutionary trade-offs in dengue virus epidemiological traits, for example, a trade-off between dengue transmission rate and its virulence to humans. Our results indicate that strategies that block transmission or reduce mosquito biting impose selection on dengue virulence in humans. This selection can be for either higher or lower virulence, depending on the interaction between the effect of the transgene and the trade-offs in epidemiological traits, highlighting the need for detailed quantitative data to understand more fully the impact of mosquito transgenesis on dengue virulence. Dengue virulence in mosquitoes can be selected on by transgenic strategies of blocking transmission, decreased mosquito biting, increased mosquito background mortality, and increased mosquito infection-induced mortality. Our results suggest that dengue control strategies that raise mosquito background mortality or mosquito infection-induced mortality pose less risk of causing increased virulence to humans than strategies that block transmission or reduce mosquito biting.

  11. Development of atopic dermatitis in mice transgenic for human apolipoprotein C1

    NARCIS (Netherlands)

    Nagelkerken, L.; Verzaal, P.; Lagerweij, T.; Persoon-Deen, C.; Berbee, J.F.P.; Prens, E.P.; Havekes, L.M.; Oranje, A.P.

    2008-01-01

    Mice with transgenic expression of human apolipoprotein C1 (APOC1) in liver and skin have strongly increased serum levels of cholesterol, triglycerides, and free fatty acids, indicative of a disturbed lipid metabolism. Importantly, these mice display a disturbed skin barrier function, evident from i

  12. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk

    OpenAIRE

    Gil, Geun-Cheol; Velander, William H.; Van Cott, Kevin E

    2008-01-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan stru...

  13. CONSTRUCTION AND STUDY OF Althaea officinalis TRANSGENIC ROOTS CULTURE WITH HUMAN INTERFERON α2B GENE

    Directory of Open Access Journals (Sweden)

    N. A. Matvieieva

    2013-04-01

    Full Text Available The aim of our work was to obtain Althaea officinalis L. «hairy» root culture with human interferon α2b gene (ifn-α2b, to measure fructans content and antiviral activity of extracts from the transgenic roots. Transformation of leaf and root explants was carried out by means of Agrobacterium rhizogenes-mediated transformation. Antiviral activity was measured by the reduction in cytopathic effect of vesicular stomatitis virus (Indiana strain in bovine kidney cells line MDBK. Transformation frequency was 100% for leaf and root explants. RT-PCR confirmed ifn- α2b gene transcription. The clones of transgenic roots differed in mass increasing from 0, 036 ± 0,008 up to 0,371 ± 0,019 g in 30 days cultivation and in fructan synthesis from 67,2± 4,47 up to 154,6 ± 6,62 mg/g roots dry weight. Extracts from «hairy»roots culture were characterized by high antiviral activity against vesicular stomatitis virus — up to 26 000 IU/ g of roots fresh weight. In some cases the genetic transformation shown to lead increasing the growth rate and increasing the level of fructan synthesis in transgenic A. officinalis roots. Extracts from cultivated in vitro marshmallow transgenic roots were characterized by high level of antiviral activity against vesicular stomatitis virus. Thus, there were obtained transgenic A. officinalis roots, characterized by high growth rate, significant accumulation of fructans and high antiviral activity.

  14. Creation of Transgenic Bananas Expressing Human Lysozyme Gene for Panama Wilt Resistance

    Institute of Scientific and Technical Information of China (English)

    Xin-Wu PEI; Shi-Kai CHEN; Rui-Ming WEN; Shang YE; Jia-Qin HUANG; Yong-Qiang ZHANG; Bing-Shan WANG; Zhi-Xing WANG; Shi-Rong JIA

    2005-01-01

    Human lysozyme (HL) inhibits Fusarium oxysporum (FocR4) growth in vitro. To obtaintransgenic bananas (Musa spp.) that are resistant to Panama wilt (F. oxysporum), we introduced an HL genethat is driven by a constitutive cauliflower mosaic virus 35S promoter into the banana via Agrobacterium-mediated transformation. PCR confirmed that 51 transgenic plants were obtained. The development ofPanama wilt symptoms were examined after the plants had been grown in pots. The non-transgenic plantsdeveloped typical fusarium symptoms 60 d after FocR4 inoculation, whereas 24 of 51 transgenic plants remained healthy. The transgenic banana plants that showed resistance to FocR4 in the pots were then planted in a field that was heavily infected with FocR4 for further investigation. Eleven of 24 plants developed symptoms before bud emergence; another 11 plants showed symptoms after bud emergence and the remaining two plants, H-67 and H-144, remained healthy and were able to fruit. Northern blotting analysisdemonstrated that H-67 and H-144, bearing the strongest resistance to Panama wilt, had the highest level ofHL expression and that the expression of HL was well correlated with the FocR4 resistance of transgenicplants. We conclude that Agrobacterium-mediated transformation, with the assistance of particlebombardment, is a powerful approach for banana transformation and that a transgenic HL gene can causeresistance of the crop to FocR4 in the field.

  15. Production of human apolipoprotein(a) transgenic NIBS miniature pigs by somatic cell nuclear transfer.

    Science.gov (United States)

    Shimatsu, Yoshiki; Horii, Wataru; Nunoya, Tetsuo; Iwata, Akira; Fan, Jianglin; Ozawa, Masayuki

    2016-01-01

    Most cases of ischemic heart disease and stroke occur as a result of atherosclerosis. The purpose of this study was to produce a new Nippon Institute for Biological Science (NIBS) miniature pig model by somatic cell nuclear transfer (SCNT) for studying atherosclerosis. The human apolipoprotein(a) (apo(a)) genes were transfected into kidney epithelial cells derived from a male and a female piglet. Male cells were used as donors initially, and 275 embryos were transferred to surrogates. Three offspring were delivered, and the production efficiency was 1.1% (3/275). Serial female cells were injected into 937 enucleated oocytes. Eight offspring were delivered (production efficiency: 0.9%) from surrogates. One male and 2 female transgenic miniature pigs matured well. Lipoprotein(a) was found in the male and one of the female transgenic animals. These results demonstrate successful production of human apo(a) transgenic NIBS miniature pigs by SCNT. Our goal is to establish a human apo(a) transgenic NIBS miniature pig colony for studying atherosclerosis.

  16. Analysis of the N-glycans of recombinant human Factor IX purified from transgenic pig milk.

    Science.gov (United States)

    Gil, Geun-Cheol; Velander, William H; Van Cott, Kevin E

    2008-07-01

    Glycosylation of recombinant proteins is of particular importance because it can play significant roles in the clinical properties of the glycoprotein. In this work, the N-glycan structures of recombinant human Factor IX (tg-FIX) produced in the transgenic pig mammary gland were determined. The majority of the N-glycans of transgenic pig-derived Factor IX (tg-FIX) are complex, bi-antennary with one or two terminal N-acetylneuraminic acid (Neu5Ac) moieties. We also found that the N-glycan structures of tg-FIX produced in the porcine mammary epithelial cells differed with respect to N-glycans from glycoproteins produced in other porcine tissues. tg-FIX contains no detectable Neu5Gc, the sialic acid commonly found in porcine glycoproteins produced in other tissues. Additionally, we were unable to detect glycans in tg-FIX that have a terminal Galalpha(1,3)Gal disaccharide sequence, which is strongly antigenic in humans. The N-glycan structures of tg-FIX are also compared to the published N-glycan structures of recombinant human glycoproteins produced in other transgenic animal species. While tg-FIX contains only complex structures, antithrombin III (goat), C1 inhibitor (rabbit), and lactoferrin (cow) have both high mannose and complex structures. Collectively, these data represent a beginning point for the future investigation of species-specific and tissue/cell-specific differences in N-glycan structures among animals used for transgenic animal bioreactors.

  17. Development of transgenic minipigs with expression of antimorphic human cryptochrome 1.

    Directory of Open Access Journals (Sweden)

    Huan Liu

    Full Text Available Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1 of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP. Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.

  18. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

    Science.gov (United States)

    Simonet, W S; DeRose, M L; Bucay, N; Nguyen, H Q; Wert, S E; Zhou, L; Ulich, T R; Thomason, A; Danilenko, D M; Whitsett, J A

    1995-01-01

    Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618921

  19. Consumption of milk from transgenic goats expressing human lysozyme in the mammary gland results in the modulation of intestinal microflora.

    Science.gov (United States)

    Maga, Elizabeth A; Walker, Richard L; Anderson, Gary B; Murray, James D

    2006-08-01

    Lysozyme is a key antimicrobial component of human milk that has several health-promoting functions including the development of a healthy intestinal tract. However, levels of lysozyme in the milk of dairy animals are negligible. We have generated transgenic dairy goats that express human lysozyme (HLZ) in their milk in an attempt to deliver the benefits of human milk in a continual fashion. To test the feasibility of this transgenic approach to achieve a biological impact at the level of the intestine, feeding trials were conducted in two animal models. Pasteurized milk from HLZ transgenic animals was fed to both kid goats (ruminant model) and young pigs (human model), and the numbers of total coliforms and Escherichia coli present in the small intestine were determined. Data from this proof-of-principle study demonstrate that milk from transgenic animals was capable of modulating the bacterial population of the gut in both animal models. Pigs that consumed pasteurized milk from HLZ transgenic goats had fewer numbers of coliforms and E. coli in their intestine than did those receiving milk from non-transgenic control animals. The opposite effect was seen in goats. Milk from these transgenic animals not only represent one of the first transgenic food products with the potential of benefiting human health, but are also a unique model to study the development and role of intestinal microflora on health, well-being and resistance to disease.

  20. The transgenic rabbit as model for human diseases and as a source of biologically active recombinant proteins.

    Science.gov (United States)

    Bosze, Zs; Hiripi, L; Carnwath, J W; Niemann, H

    2003-10-01

    Until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis; however, progress in somatic cell cloning based on nuclear transfer will soon make it possible to produce rabbits with modifications to specific genes by the combination of homologous recombination and subsequent prescreening of nuclear donor cells. Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. Transgenic rabbits have also proved to be suitable bioreactors for the production of recombinant protein both on an experimental and a commercial scale. This review summarizes recent research based on the transgenic rabbit model.

  1. Transgenic expression of the human growth hormone minigene promotes pancreatic β-cell proliferation.

    Science.gov (United States)

    Baan, Mieke; Kibbe, Carly R; Bushkofsky, Justin R; Harris, Ted W; Sherman, Dawn S; Davis, Dawn Belt

    2015-10-01

    Transgenic mouse models are designed to study the role of specific proteins. To increase transgene expression the human growth hormone (hGH) minigene, including introns, has been included in many transgenic constructs. Until recently, it was thought that the hGH gene was not spliced, transcribed, and translated to produce functional hGH protein. We generated a transgenic mouse with the transcription factor Forkhead box M1 (FoxM1) followed by the hGH minigene, under control of the mouse insulin promoter (MIP) to target expression specifically in the pancreatic β-cell. Expression of FoxM1 in isolated pancreatic islets in vitro stimulates β-cell proliferation. We aimed to investigate the effect of FoxM1 on β-cell mass in a mouse model for diabetes mellitus. However, we found inadvertent coexpression of hGH protein from a spliced, bicistronic mRNA. MIP-FoxM1-hGH mice had lower blood glucose and higher pancreatic insulin content, due to increased β-cell proliferation. hGH signals through the murine prolactin receptor, and expression of its downstream targets tryptophan hydroxylase-1 (Tph1), tryptophan hydroxylase-2 (Tph2), and cytokine-inducible SH2 containing protein (Cish) was increased. Conversely, transcriptional targets of FoxM1 were not upregulated. Our data suggest that the phenotype of MIP-FoxM1-hGH mice is due primarily to hGH activity and that the FoxM1 protein remains largely inactive. Over the past decades, multiple transgenic mouse strains were generated that make use of the hGH minigene to increase transgene expression. Our work suggests that each will need to be carefully screened for inadvertent hGH production and critically evaluated for the use of proper controls.

  2. Stable Skin-specific Overexpression of Human CTLA4-Ig in Transgenic Mice through Seven Generations

    Institute of Scientific and Technical Information of China (English)

    Yong WANG; Yong NI; Hong WEI; Feng-Chao WANG; Liang-Peng GE; Xiang GAO

    2006-01-01

    Skin graft rejection is a typical cellular immune response, mainly mediated by T cells. Cytotoxic T lymphocyte associated antigen 4-immunoglobin (CTLA4-Ig) extends graft survival by blocking the T cell co-stimulation pathway and inhibiting T cell activation. To investigate the efficacy of CTLA4-Ig in prolonging skin graft survival, human CTLA4-Ig (hCTLA4-Ig) was engineered to overexpress in mouse skin by transgenesis using the K14 promoter. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay indicated that the expression of CTLA4-Ig remained skin-specific and relatively constant compared to the internal control protein, AKT, through seven generations. The presence and concentration of the hCTLA4-Ig protein in transgenic mouse sera was determined by enzyme-linked immunosorbent assay (ELISA), and the results indicated that the serum CTLA4-Ig concentration also remained constant through generations. Survival of transgenic mouse skins grafted onto rat wounds was remarkably prolonged compared to that of wild-type skins from the same mouse strain, and remained comparable among all seven generations. This suggested that the bioactive hCTLA4-Ig protein was stably expressed in transgenical mice through at least seven generations, which was consistent with the stable skin-specific CTLA4-Ig expression.The results demonstrated that the transgenic expression of hCTLA4-Ig in skin driven by the K14 promoter remained constant through generations, and a transgenic line can be established to provide transgenic skin with extended survival reproducibly.

  3. Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

    Directory of Open Access Journals (Sweden)

    Chan Anthony WS

    2008-02-01

    Full Text Available Abstract Background Alzheimer's disease (AD is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD. Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP. Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. Results Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. Conclusion This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue

  4. A proteomic study to identify soya allergens--the human response to transgenic versus non-transgenic soya samples.

    Science.gov (United States)

    Batista, Rita; Martins, Isabel; Jeno, Paul; Ricardo, Cândido Pinto; Oliveira, Maria Margarida

    2007-01-01

    In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya versus its non-transgenic control. We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples. We identified 2 new potential soybean allergens--one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study. Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment. Copyright (c) 2007 S. Karger AG, Basel.

  5. Expression of human transferrin can be regulated effectively by rabbit transferrin regulatory elements in transgenic mice.

    Science.gov (United States)

    Yan, Jingbin; Gong, Xiuli; Pan, Shubiao; Guo, Xinbing; Ren, Zhaorui; Zeng, Yitao

    2014-06-01

    Human transferrin (hTF) belongs to the iron-binding glycoprotein family. It plays an important role in iron transport throughout the body. Transgenic mice are a good model to study how to produce functional hTF on a large-scale. We have improved the expression of hTF and investigated its regulatory mechanism in transgenic mice. Three expression constructs were prepared in which hTF expression was controlled by different regulatory cassettes of rabbit transferrin (rTF). hTF was secreted into serum of transgenic mice when its expression was controlled by the rTF promoter and enhancer, whereas the rTF enhancer in tandem with the rTF promoter repressed hTF secretion into milk. A significant inverse relationship between methylation of the rTF promoter and hTF expression was observed in liver, heart, mammary gland, and muscle of transgenic mice. The highest concentration of hTF was 700 μg/ml in milk.

  6. Human anti-rhesus D IgG1 antibody produced in transgenic plants

    DEFF Research Database (Denmark)

    Bouquin, Thomas; Thomsen, Mads; Nielsen, Leif Kofoed

    2002-01-01

    antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells. Plant-derived antibody had equivalent......Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D...... properties to CHO cell-produced anti-RhD antibody, indicating its potential usefulness in diagnostic and therapeutic programs....

  7. Regulation of human clotting factor IX cDNA expression in transgenic mice

    Institute of Scientific and Technical Information of China (English)

    胡以平; 邱信芳; 薛京伦; 刘祖洞

    1995-01-01

    To study the expression of human dotting factor IX cDNA in transgenic mice,Which is an es-sential work on gene therapy for hemophilia B,3 recombinant constructions containing different lengths ofhuman dotting factor IX cDNA have been introduced into the cultured cells.All of the recombinant constructionswere found to he expressed well in vitro.They were then microinjected into the male pronudei of the fertilizedmouse eggs respectively for generating trahsgenic mice.Unfortunately,none of them was expressed in any transgenicmice.These results show that the expression of the human clotting factor IX cDNA in the transgenic mice canbe determined by cis regulatory element(s).As compared With the results from other related works,it is sug-gested that the cis regulatory element(s)is resided in the 5’-end non-coding region.

  8. Free Access to Running Wheels Abolishes Hyperphagia in Human Growth Hormone Transgenic Rats

    OpenAIRE

    KOMATSUDA, Mugiko; Yamanouchi, Keitaro; Matsuwaki, Takashi; Nishihara, Masugi

    2014-01-01

    ABSTRACT Obesity is a major health problem, and increased food intake and decreased physical activity are considered as two major factors causing obesity. Previous studies show that voluntary exercise in a running wheel decreases not only body weight but also food intake of rats. We previously produced human growth hormone transgenic (TG) rats, which are characterized by severe hyperphagia and obesity. To gain more insight into the effects on physical activity to food consumption and obesity,...

  9. Pressure-natriuresis and -diuresis in transgenic rats harboring both human renin and human angiotensinogen genes.

    Science.gov (United States)

    Dehmel, B; Mervaala, E; Lippoldt, A; Gross, V; Bohlender, J; Ganten, D; Luft, F C

    1998-12-01

    The hypertensive double transgenic rat harboring both the human renin and human angiotensinogen genes (dTGR) offers a unique opportunity to study the human renin-angiotensin system in an experimental animal model. Since nothing is known about the control of sodium and water excretion in these rats, this study was performed to compare pressure-natriuresis relationships in hypertensive dTGR and normotensive control rats harboring only the human renin gene (hREN), in order to determine how the pressure-natriuresis relationship is reset in hypertensive dTGR. To differentiate between extrinsic and intrinsic renal mechanisms, experiments were performed with and without renal denervation, and with and without infusions of vasopressin, norepinephrine, 17-OH-corticosterone, and aldosterone. Human and rat angiotensinogen and renin mRNA expression were also determined. In hREN without controlled renal function, urine flow and sodium excretion increased from 13 to 169 microl/min per g kidney wet weight (kwt) and from 1 to 30 micromol/min per g kwt, respectively, as renal perfusion pressure was increased from 67 to 135 mmHg. Renal blood flow (RBF) and GFR ranged between 3 to 7 and 0.9 to 1.5 ml/min per g kwt. In dTGR, pressure-natriuresis-diuresis relationships were shifted approximately 40 mmHg rightward. RBF was lower in dTGR than in hREN; GFR was not different. In dTGR with neurohormonal factors controlled, RBF was decreased and pressure-natriuresis-diuresis curves were not different compared to dTGR curves without these interventions. By light microscopy, the kidneys of these 6-wk-old dTGR and hREN rats were normal and indistinguishable. Both human and rat renin and angiotensinogen mRNA were expressed in the kidneys of dTGR. The two renin mRNA were decreased in dTGR, indicating a physiologic downregulation of renin gene expression by high BP. It is concluded that the renal pressure-natriuresis mechanism is reset toward higher pressure levels in dTGR and participates in the

  10. Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    Qiuyan Li; Hengxi Wei; Ying Guo; Yan Li; Rui Zhao; Yufang Ma; Zhengquan Yu; Bo Tang; Lei Zhang; Yunping Dai; Ning Li

    2009-01-01

    Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, s1w3 and s1w6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after acti-vation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.

  11. Transgenic mice designed to express human α-1,2-fucosyltransferase in combination of human DAF and CD59 to avoid xenograft rejection

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The expression of human α-1,2-fucosyltransferase (HT) or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation. In this study, we examined whether peripheral blood mononuclear cells (PBMCs) from polytransgenic mice expressing the human HT, and complement regulatory proteins (DAF and CD59), can provide more effective protection against xenograft rejection. Transgenic mice were produced by co-injection of gene constructs for human HT, DAF and/or CD59. Flow Cytometry (FCM) was used to screen the positive transgenic mice. PBMCs from transgenic mice were incubated with 15% human serum to evaluate natural antibody binding, complement activation and expression of adhesion molecules. Three transgenes were strongly expressed in PBMCs of transgenic mice, and HT expression signifi- cantly reduced expression of the major xenoepitope galactose-α-1,3-galactose (α-Gal). Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their re- sistance to human serum-mediated cytolysis when compared with single transgenic PBMCs. Moreover, the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolysis, avoided hyperacute rejection and reduced expression of adhesion mole- cules. Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection. The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.

  12. Transgenic mice designed to express human α-1,2-fucosyltransferase in combination of human DAF and CD59 to avoid xenograft rejection

    Institute of Scientific and Technical Information of China (English)

    LIU BingQian; CHENG ChuanYu; WU YuDong; WEI JinXing; LI GuangSan; MA TengXiang

    2008-01-01

    The expression of human α-1,2-fucosyltransferase (HT) or complement regulatory proteins has been proved as an strategy to overcome hypercute rejection in discordant xenogeneic organ transplantation.In this study, we examined whether peripheral blood mononuclear cells (PBMCs) from polytransgenic mice expressing the human HT, and complement regulatory proteins (DAF and CD59), can provide more effective protection against xenograft rejection. Transgenic mice were produced by co-injection of gene constructs for human HT, DAF and/or CD59. Flow Cytometry (FCM) was used to screen the positive transgenic mice. PBMCs from transgenic mice were incubated with 15% human serum to evaluate natural antibody binding, complement activation and expression of adhesion molecules.Three transgenes were strongly expressed in PBMCs of transgenic mice, and HT expression significantly reduced expression of the major xenoepitope galactose-α-1,3-galactose (α-Gal). Functional studies with PBMCs showed that co-expression of HT and DAF or CD59 markedly increased their resistance to human serum-mediated cytolysis when compared with single transgenic PBMCs. Moreover,the combined expression of triple transgenes in PBMCs led to the greatest protection against human serum-mediated cytolyais, avoided hyperacute rejection and reduced expression of adhesion molecules. Strong co-expression of triple transgenes was completely protected from xenograft hyperacute rejection and partially inhibited acute vascular rejection. The studies suggest that engineering mice to express triple molecules represents an critical step toward prolonging xenograft survival and might be more suitable for xenotransplantation.

  13. Human apoB contributes to increased serum total apo(a level in LPA transgenic mice

    Directory of Open Access Journals (Sweden)

    Teivainen Päivi A

    2004-05-01

    Full Text Available Abstract Background The Lp(a lipoprotein (Lp(a consists of the polymorphic glycoprotein apolipoprotein(a (apo(a, which is attached by a disulfide bond to apolipoprotein B (apoB. Apo(a, which has high homology with plasminogen, is present only in primates and hedgehogs. However, transgenic mice and rabbits with high serum apo(a levels exist. Liver is the main site for apo(a synthesis, but the site of removal is uncertain. To examine differences between transgenic mice expressing the LPA gene and mice capable of forming Lp(a particles, LPA-YAC transgenic mice and hAPOB transgenic mice were crossed and their offspring examined. Results Comparison of LPA-YAC with LPA-YAC/hAPOB transgenic mice showed that LPA-YAC/hAPOB transgenic mice have higher serum total apo(a and total cholesterol level than mice lacking the hAPOB gene. However, hepatic apo(a mRNA level was higher in LPA-YAC transgenic mice than in LPA-YAC/hAPOB transgenic mice. Feeding of a high-cholesterol/high-fat diet to male LPA-YAC transgenic mice with or without the hAPOB gene resulted in reduced serum total apo(a and hepatic apo(a mRNA level. Conclusion In conclusion, the higher serum total apo(a level in LPA-YAC/hAPOB transgenic mice than in LPA-YAC transgenic mice is not caused by increased apo(a synthesis. Lower hepatic apo(a mRNA level in LPA-YAC/hAPOB than in LPA-YAC transgenic mice may suggest that the increase in total apo(a level is a result of apo(a accumulation in serum. Furthermore, observed higher serum total cholesterol level in LPA-YAC/hAPOB transgenic mice than either in wild type or LPA-YAC transgenic mice may further suggest that human APOB transgenicity is a factor that contributes to increased serum total apo(a and cholesterol levels. Our results on reduced serum total apo(a and hepatic apo(a mRNA levels in HCHF fed male LPA-YAC transgenic mice confirm earlier findings in females, and show that there are no sex difference in mechanisms for lowering apo(a level in

  14. Bridging the species divide: transgenic mice humanized for type-I interferon response.

    Directory of Open Access Journals (Sweden)

    Daniel Harari

    Full Text Available We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs enabling the study of type I human interferons (Hu-IFN-Is in mice. These "HyBNAR" (Hybrid IFNAR mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2. HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase. Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.

  15. Progress Toward a Human CD4/CCR5 Transgenic Rat Model for De Novo Infection by Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Keppler, Oliver T.; Welte, Frank J.; Ngo, Tuan A.; Chin, Peggy S.; Patton, Kathryn S.; Tsou, Chia-Lin; Abbey, Nancy W.; Sharkey, Mark E.; Grant, Robert M.; You, Yun; Scarborough, John D.; Ellmeier, Wilfried; Littman, Dan R.; Stevenson, Mario; Charo, Israel F.; Herndier, Brian G.; Speck, Roberto F.; Goldsmith, Mark A.

    2002-01-01

    The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4+ T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4+ T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2–long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection. PMID:11901198

  16. Ethical issues of transplanting organs from transgenic animals into human beings.

    Science.gov (United States)

    Behnam Manesh, Shima; Omani Samani, Reza; Behnam Manesh, Shayan

    2014-01-01

    One of the most important applications of transgenic animals for medical purposes is to transplant their organs into human's body, an issue which has caused a lot of ethical and scientific discussions. we can divide the ethical arguments to two comprehensive groups; the first group which is known as deontological critiques (related to the action itself regardless of any results pointing the human or animal) and the second group, called the consequentialist critiques (which are directly pointing the consequences of the action). The latter arguments also can be divided to two subgroups. In the first one which named anthropocentrism, just humankind has inherent value in the moral society, and it studies the problem just from a human-based point of view while in second named, biocentrism all the living organism have this value and it deals specially with the problem from the animal-based viewpoint. In this descriptive-analytic study, ethical issues were retrieved from books, papers, international guidelines, thesis, declarations and instructions, and even some weekly journals using keywords related to transgenic animals, organ, and transplantation. According to the precautionary principle with the strong legal and ethical background, due to lack of accepted scientific certainties about the safety of the procedure, in this phase, transplanting animal's organs into human beings have the potential harm and danger for both human and animals, and application of this procedure is unethical until the safety to human will be proven.

  17. Doxycycline-Induced Expression of Transgenic Human Tumor Necrosis Factor α in Adult Mice Results in Psoriasis-like Arthritis

    Science.gov (United States)

    Retser, Eugen; Schied, Tanja; Skryabin, Boris V; Vogl, Thomas; Kanczler, Janos M; Hamann, Nina; Niehoff, Anja; Hermann, Sven; Eisenblätter, Michel; Wachsmuth, Lydia; Pap, Thomas; van Lent, Peter L E M; Loser, Karin; Roth, Johannes; Zaucke, Frank; Ludwig, Stephan; Wixler, Viktor

    2013-01-01

    Objective To generate doxycycline-inducible human tumor necrosis factor α (TNFα)–transgenic mice to overcome a major disadvantage of existing transgenic mice with constitutive expression of TNFα, which is the limitation in crossing them with various knockout or transgenic mice. Methods A transgenic mouse line that expresses the human TNFα cytokine exclusively after doxycycline administration was generated and analyzed for the onset of diseases. Results Doxycycline-inducible human TNFα–transgenic mice developed an inflammatory arthritis– and psoriasis-like phenotype, with fore and hind paws being prominently affected. The formation of “sausage digits” with characteristic involvement of the distal interphalangeal joints and nail malformation was observed. Synovial hyperplasia, enthesitis, cartilage and bone alterations, formation of pannus tissue, and inflammation of the skin epidermis and nail matrix appeared as early as 1 week after the treatment of mice with doxycycline and became aggravated over time. The abrogation of human TNFα expression by the removal of doxycycline 6 weeks after beginning stimulation resulted in fast resolution of the most advanced macroscopic and histologic disorders, and 3–6 weeks later, only minimal signs of disease were visible. Conclusion Upon doxycycline administration, the doxycycline-inducible human TNFα–transgenic mouse displays the major features of inflammatory arthritis. It represents a unique animal model for studying the molecular mechanisms of arthritis, especially the early phases of disease genesis and tissue remodeling steps upon abrogation of TNFα expression. Furthermore, unlimited crossing of doxycycline-inducible human TNFα–transgenic mice with various knockout or transgenic mice opens new possibilities for unraveling the role of various signaling molecules acting in concert with TNFα. PMID:23740547

  18. Ethical Issues of Transplanting Organs from Transgenic Animals into Human Beings

    Directory of Open Access Journals (Sweden)

    Shima Behnam Manesh

    2014-04-01

    Full Text Available One of the most important applications of transgenic animals for medical purposes is to transplant their organs into human’s body, an issue which has caused a lot of ethical and scientific discussions. we can divide the ethical arguments to two comprehensive groups; the first group which is known as deontological critiques (related to the action itself regardless of any results pointing the human or animal and the second group, called the consequentialist critiques (which are directly pointing the consequences of the action. The latter arguments also can be divided to two subgroups. In the first one which named anthropocentrism, just humankind has inherent value in the moral society, and it studies the problem just from a human-based point of view while in second named, biocentrism all the living organism have this value and it deals specially with the problem from the animal-based viewpoint. In this descriptive-analytic study, ethical issues were retrieved from books, papers, international guidelines, thesis, declarations and instructions, and even some weekly journals using keywords related to transgenic animals, organ, and transplantation. According to the precautionary principle with the strong legal and ethical background, due to lack of accepted scientific certainties about the safety of the procedure, in this phase, transplanting animal’s organs into human beings have the potential harm and danger for both human and animals, and application of this procedure is unethical until the safety to human will be proven.

  19. MHC class I phenotype and function of human beta 2-microglobulin transgenic murine lymphocytes

    DEFF Research Database (Denmark)

    Bjerager, L; Pedersen, L O; Bregenholt, S;

    1996-01-01

    Lymphoid cells from beta 2-microglobulin (beta 2m) knockout mice transgenic for human (h) beta 2m (C57BL/10 m beta 2m-/h beta 2m+) were compared with normal mice for their binding to exogenously added h beta 2m, binding to a H-2Db peptide and for functional activity in a one-way allogenic MLC....... Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous h beta 2m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/h beta 2m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS...... binds radiolabelled peptide in the absence of exogenous added h beta 2m suggesting that a stable fraction of hybrid H-2Db molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than...

  20. Expression of human coagulation Factor IX in transgenic tomato (Lycopersicon esculentum).

    Science.gov (United States)

    Zhang, Hui; Zhao, Lingxia; Chen, Yuhui; Cui, Lijie; Ren, Weiwei; Tang, Kexuan

    2007-10-01

    In the present study, a plant binary expression vector PG-pRD12-hFIX (where PG is polygalacturonase) harbouring the hFIX (human coagulation Factor IX) gene was constructed and introduced into tomato (Lycopersicon esculentum) via Agrobacterium tumefaciens-mediated transformation. After kanamycin selection, 32 putative independent transgenic tomato plants were regenerated. PCR and Southern-blot analyses confirmed the transgenic status of some plants. RT (reverse transcription)-PCR analysis for the expression of the introduced gene (hFIX) demonstrated that the hFIX gene was expressed specifically in fruits of the tomato. Western-blot analysis confirmed the presence of a 56 kDa band specific to hFIX in the transformed tomatoes. ELISA results showed that the expression of hFIX protein reached a maximum of 15.84 ng/g fresh weight in mature fruit. A blood-clotting assay demonstrated the clotting activity of the expressed hFIX protein in transgenic tomato fruits. This is the first report on the expression of hFIX in plants, and our research provides potentially valuable knowledge for further development of the plant-derived therapeutic proteins.

  1. HLA-B27 and human β2-microglobulin affect the gut microbiota of transgenic rats.

    Directory of Open Access Journals (Sweden)

    Phoebe Lin

    Full Text Available The HLA-B27 gene is a major risk factor for clinical diseases including ankylosing spondylitis, acute anterior uveitis, reactive arthritis, and psoriatic arthritis, but its mechanism of risk enhancement is not completely understood. The gut microbiome has recently been shown to influence several HLA-linked diseases. However, the role of HLA-B27 in shaping the gut microbiome has not been previously investigated. In this study, we characterize the differences in the gut microbiota mediated by the presence of the HLA-B27 gene. We identified differences in the cecal microbiota of Lewis rats transgenic for HLA-B27 and human β2-microglobulin (hβ2m, compared with wild-type Lewis rats, using biome representational in situ karyotyping (BRISK and 16S rRNA gene sequencing. 16S sequencing revealed significant differences between transgenic animals and wild type animals by principal coordinates analysis. Further analysis of the data set revealed an increase in Prevotella spp. and a decrease in Rikenellaceae relative abundance in the transgenic animals compared to the wild type animals. By BRISK analysis, species-specific differences included an increase in Bacteroides vulgatus abundance in HLA-B27/hβ2m and hβ2m compared to wild type rats. The finding that HLA-B27 is associated with altered cecal microbiota has not been shown before and can potentially provide a better understanding of the clinical diseases associated with this gene.

  2. Correction of murine mucopolysaccharidosis VII by a human. beta. -glucuronidase transgene

    Energy Technology Data Exchange (ETDEWEB)

    Kyle, J.W.; Vogler, C.; Hoffmann, J.W.; Sly, W.S. (St. Louis Univ. School of Medicine, MO (USA)); Birkenmeier, E.H.; Gwynn, B. (Jackson Laboratory, Bar Harbor, ME (USA))

    1990-05-01

    The authors recently described a murine model for mucopolysaccharidosis VII in mice that have an inherited deficiency of {beta}-glucuronidase. Affected mice, of genotype gus{sup mps}/gus{sup mps}, present clinical manifestations similar to those of humans with mucopolysaccharidosis VII (Sly syndrome) and are shown here to have secondary elevations of other lysosomal enzymes. The mucopolysaccharidosis VII phenotype in both species includes dwarfism, skeletal deformities, and premature death. Lysosome storage is visualized within enlarged vesicles and correlates biochemically with accumulation of undegraded and partially degraded glycosaminoglycans. In this report they describe the consequences of introducing the human {beta}-glucuronidase gene, GUSB, into gus{sup mps}/gus{sup mps} mice that produce virtually no murine {beta}-glucuronidase. Transgenic mice homozygous for the mucopolysaccharidosis VII mutation expressed high levels of human {beta}-glucuronidase activity in all tissues examined and were phenotypically normal. Biochemically, both the intralysosomal storage of glycosaminoglycans and the secondary elevation of other acid hydrolases were corrected. These findings demonstrate that the GUSB transgene is expressed in gus{sup mps}/gus{sup mps} mice and that human {beta}-glucuronidase corrects the murine mucopolysaccharidosis storage disease.

  3. Using transgenic plants and modified plant viruses for the development of treatments for human diseases.

    Science.gov (United States)

    Loh, Hwei-San; Green, Brian J; Yusibov, Vidadi

    2017-08-08

    Production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. A major milestone for plant-based protein production for use in human health was achieved when Protalix BioTherapeutics produced taliglucerase alfa (Elelyso(®)) in suspension cultures of a transgenic carrot cell line for the treatment of patients with Gaucher's disease, was approved by the USA Food and Drug Administration in 2012. In this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice

    Directory of Open Access Journals (Sweden)

    Palm Kaia

    2009-06-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF is a small secreted protein that has important roles in the developing and adult nervous system. Altered expression or changes in the regulation of the BDNF gene have been implicated in a variety of human nervous system disorders. Although regulation of the rodent BDNF gene has been extensively investigated, in vivo studies regarding the human BDNF gene are largely limited to postmortem analysis. Bacterial artificial chromosome (BAC transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. Results In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of the human BDNF locus modified such that BDNF coding sequence was replaced with the sequence of a fusion protein consisting of N-terminal BDNF and the enhanced green fluorescent protein (EGFP. The human BDNF-BAC construct containing all BDNF 5' exons preceded by different promoters recapitulated the expression of endogenous BDNF mRNA in the brain and several non-neural tissues of transgenic mice. All different 5' exon-specific BDNF-EGFP alternative transcripts were expressed from the transgenic human BDNF-BAC construct, resembling the expression of endogenous BDNF. Furthermore, BDNF-EGFP mRNA was induced upon treatment with kainic acid in a promotor-specific manner, similarly to that of the endogenous mouse BDNF mRNA. Conclusion Genomic region covering 67 kb of human BDNF gene, 84 kb of upstream and 17 kb of downstream sequences is sufficient to drive tissue-specific and kainic acid-induced expression of the reporter gene in transgenic mice. The pattern of expression of the transgene is highly similar to BDNF gene expression in mouse and human. This is the first study to show that human BDNF gene is regulated by neural activity.

  5. Immune status following alemtuzumab treatment in human CD52 transgenic mice.

    Science.gov (United States)

    Turner, Michael J; Lamorte, Michael J; Chretien, Nathalie; Havari, Evis; Roberts, Bruce L; Kaplan, Johanne M; Siders, William M

    2013-08-15

    Alemtuzumab is a monoclonal antibody against the CD52 antigen present at high levels on the surface of lymphocytes. While treatment of multiple sclerosis patients with alemtuzumab results in marked depletion of lymphocytes from the circulation, it has not been associated with a high incidence of serious infections. In a human CD52 transgenic mouse, alemtuzumab treatment showed minimal impact on the number and function of innate immune cells. A transient decrease in primary adaptive immune responses was observed post-alemtuzumab but there was little effect on memory responses. These results potentially help explain the level of immunocompetence observed in alemtuzumab-treated MS patients.

  6. Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

    OpenAIRE

    Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

    2012-01-01

    The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as wel...

  7. Role of papillomavirus oncogenes in human cervical cancer: Transgenic animal studies

    Energy Technology Data Exchange (ETDEWEB)

    Griep, A.E.; Lambert, P.F. [Univ. of Wisconsin School of Medicine, Madison, WI (United States)

    1994-05-01

    Human papillomaviruses are believed to be etiologic agents for the majority of human cervical carcinoma, a common cancer that is a leading cause of death by cancer among women worldwide. In cervical carcinoma, a subset of papillomaviral genes, namely E6 and E7, are expressed. In vitro tissue culture studies indicate that HPV E6 and E7 are oncogenes, and that their oncogenicity is due in part to their capacity to inactivate cellular tumor suppressor genes. The behavior of E6 and E7 in vitro and the genetic evidence from analysis of human cancers suggest that the E6 and E7 genes play a significant role in the development of cervical cancer. This hypothesis is now being tested using animal models. In this review, we summarize our current knowledge of the oncogenicity of papillomavirus genes that has been generated through their study in transgenic mice. 82 refs., 4 figs., 1 tab.

  8. Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John

    2013-11-01

    Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

  9. Chymase activities and survival in endotoxin-induced human chymase transgenic mice.

    Science.gov (United States)

    Rafiq, Kazi; Fan, Yu-Yan; Sherajee, Shamshad J; Takahashi, Yoshimasa; Matsuura, Junji; Hase, Naoki; Mori, Hirohito; Nakano, Daisuke; Kobara, Hideki; Hitomi, Hirofumi; Masaki, Tsutomu; Urata, Hidenori; Nishiyama, Akira

    2014-01-01

    We examined the effects of overexpressed human chymase on survival and activity in lipopolysaccharide (LPS)-treated mice. Human chymase transgenic (Tg) and wild-type C57BL/6 (WT) mice were treated with LPS (0.03, 0.1 and 0.3 mg/day; intraperitoneal) for 2 weeks. Treatment with 0.03 mg LPS did not affect survival in either WT or Tg mice. WT mice were not affected by 0.1 mg/day of LPS, whereas 25% of Tg mice died. Survival of mice treated with 0.3 mg/day of LPS was 87.5% and 0% in WT and Tg, respectively. LPS-induced increases in chymase activity in the heart and skin were significantly greater in Tg than WT mice. These data suggest a possible contribution of human chymase activation to LPS-induced mortality.

  10. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

    OpenAIRE

    Fang Zeng; Zicong Li; Qingchun Zhu; Rui Dong; Chengcheng Zhao; Guoling Li; Guo Li; Wenchao Gao; Gelong Jiang; Enqin Zheng; Gengyuan Cai; Stefan Moisyadi; Johann Urschitz; Huaqiang Yang; Dewu Liu

    2017-01-01

    The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μ...

  11. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  12. Age-associated and cell-type-specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit.

    Science.gov (United States)

    Vickers, J C; Morrison, J H; Friedrich, V L; Elder, G A; Perl, D P; Katz, R N; Lazzarini, R A

    1994-09-01

    Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2-25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.

  13. High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

    Science.gov (United States)

    Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos

    2006-07-13

    Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

  14. Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter

    Institute of Scientific and Technical Information of China (English)

    VerenaNordhoff; JorgGromoll; LucaFoppiani; C.MarcLuetjens; StefanSchlatt; ElenaKostova; IlpoHuhtaniemi; EberhardNieschlag; ManuelaSimoni

    2003-01-01

    Aim:To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function.Methods:We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5’-flanking region fragment of its promoter.Results: Mice were phenotypically normal and fertile.In males,mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic littermates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells,spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5"-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

  15. Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies.

    Science.gov (United States)

    Zhu, Hao; Shah, Samar; Shyh-Chang, Ng; Shinoda, Gen; Einhorn, William S; Viswanathan, Srinivas R; Takeuchi, Ayumu; Grasemann, Corinna; Rinn, John L; Lopez, Mary F; Hirschhorn, Joel N; Palmert, Mark R; Daley, George Q

    2010-07-01

    Recently, genome-wide association studies have implicated the human LIN28B locus in regulating height and the timing of menarche. LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators of mammalian body size and metabolism. To explore the function of the Lin28-Let-7 pathway in vivo, we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. Investigation of metabolic and endocrine mechanisms of overgrowth in these transgenic mice revealed increased glucose metabolism and insulin sensitivity. Here we report a mouse that models the human phenotypes associated with genetic variation in the Lin28-Let-7 pathway.

  16. Generation of human MHC (HLA-A11/DR1) transgenic mice for vaccine evaluation

    Science.gov (United States)

    Zeng, Yang; Gao, Tongtong; Zhao, Guangyu; Jiang, Yuting; Yang, Yi; Yu, Hong; Kou, Zhihua; Lone, Yuchun; Sun, Shihui; Zhou, Yusen

    2016-01-01

    ABSTRACT The rapid occurrence of emerging infectious diseases demonstrates an urgent need for a new preclinical experimental model that reliably replicates human immune responses. Here, a new homozygous humanized human leukocyte antigen (HLA)-A11/DR1 transgenic mouse (HLA-A11+/+/DR01+/+/H-2-β2m−/−/IAβ−/−) was generated by crossing HLA-A11 transgenic (Tg) mice with HLA-A2+/+/DR01+/+/H-2-β2m−/−/IAβ−/− mice. The HLA-A11-restricted immune response of this mouse model was then examined. HLA-A11 Tg mice expressing a chimeric major histocompatibility complex (MHC) molecule comprising the α1, α2, and β2m domains of human HLA-A11 and the α3 transmembrane and cytoplasmic domains of murine H-2Db were generated. The correct integration of HLA-A11 and HLA-DR1 into the genome of the HLA-A11/DR1 Tg mice (which lacked the expression of endogenous H-2-I/II molecules) was then confirmed. Immunizing mice with a recombinant HBV vaccine or a recombinant HIV-1 protein resulted in the generation of IFN-γ-producing cytotoxic T lymphocyte (CTL) and antigen-specific antibodies. The HLA-A11-restricted CTL response was directed at HLA immunodominant epitopes. These mice represent a versatile animal model for studying the immunogenicity of HLA CTL epitopes in the absence of a murine MHC response. The established animal model will also be useful for evaluating and optimizing T cell-based vaccines and for studying differences in antigen processing between mice and humans. PMID:26479036

  17. The CD3 versus CD7 plot in multicolor flow cytometry reflects progression of disease stage in patients infected with HTLV-I.

    Directory of Open Access Journals (Sweden)

    Seiichiro Kobayashi

    Full Text Available PURPOSE: In a recent study to purify adult T-cell leukemia-lymphoma (ATL cells from acute-type patients by flow cytometry, three subpopulations were observed in a CD3 versus CD7 plot (H: CD3(highCD7(high; D: CD3(dimCD7(dim; L: CD3(dimCD7(low. The majority of leukemia cells were enriched in the L subpopulation and the same clone was included in the D and L subpopulations, suggesting clonal evolution. In this study, we analyzed patients with indolent-type ATL and human T-cell leukemia virus type I (HTLV-I asymptomatic carriers (ACs to see whether the CD3 versus CD7 profile reflected progression in the properties of HTLV-I-infected cells. EXPERIMENTAL DESIGN: Using peripheral blood mononuclear cells from patient samples, we performed multi-color flow cytometry. Cells that underwent fluorescence-activated cell sorting were subjected to molecular analyses, including inverse long PCR. RESULTS: In the D(% versus L(% plot, patient data could largely be categorized into three groups (Group 1: AC; Group 2: smoldering- and chronic-type ATL; and Group 3: acute-type ATL. Some exceptions, however, were noted (e.g., ACs in Group 2. In the follow-up of some patients, clinical disease progression correlated well with the CD3 versus CD7 profile. In clonality analysis, we clearly detected a major clone in the D and L subpopulations in ATL cases and, intriguingly, in some ACs in Group 2. CONCLUSION: We propose that the CD3 versus CD7 plot reflects progression of disease stage in patients infected with HTLV-I. The CD3 versus CD7 profile will be a new indicator, along with high proviral load, for HTLV-I ACs in forecasting disease progression.

  18. Xmrk, kras and myc transgenic zebrafish liver cancer models share molecular signatures with subsets of human hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Weiling Zheng

    Full Text Available Previously three oncogene transgenic zebrafish lines with inducible expression of xmrk, kras or Myc in the liver have been generated and these transgenic lines develop oncogene-addicted liver tumors upon chemical induction. In the current study, comparative transcriptomic approaches were used to examine the correlation of the three induced transgenic liver cancers with human liver cancers. RNA profiles from the three zebrafish tumors indicated relatively small overlaps of significantly deregulated genes and biological pathways. Nevertheless, the three transgenic tumor signatures all showed significant correlation with advanced or very advanced human hepatocellular carcinoma (HCC. Interestingly, molecular signature from each oncogene-induced zebrafish liver tumor correlated with only a small subset of human HCC samples (24-29% and there were conserved up-regulated pathways between the zebrafish and correlated human HCC subgroup. The three zebrafish liver cancer models together represented nearly half (47.2% of human HCCs while some human HCCs showed significant correlation with more than one signature defined from the three oncogene-addicted zebrafish tumors. In contrast, commonly deregulated genes (21 up and 16 down in the three zebrafish tumor models generally showed accordant deregulation in the majority of human HCCs, suggesting that these genes might be more consistently deregulated in a broad range of human HCCs with different molecular mechanisms and thus serve as common diagnosis markers and therapeutic targets. Thus, these transgenic zebrafish models with well-defined oncogene-induced tumors are valuable tools for molecular classification of human HCCs and for understanding of molecular drivers in hepatocarcinogenesis in each human HCC subgroup.

  19. Synaptotrophic effects of human amyloid beta protein precursors in the cortex of transgenic mice.

    Science.gov (United States)

    Mucke, L; Masliah, E; Johnson, W B; Ruppe, M D; Alford, M; Rockenstein, E M; Forss-Petter, S; Pietropaolo, M; Mallory, M; Abraham, C R

    1994-12-15

    The amyloid precursor protein (APP) is involved in Alzheimer's disease (AD) because its degradation products accumulate abnormally in AD brains and APP mutations are associated with early onset AD. However, its role in health and disease appears to be complex, with different APP derivatives showing either neurotoxic or neurotrophic effects in vitro. To elucidate the effects APP has on the brain in vivo, cDNAs encoding different forms of human APP (hAPP) were placed downstream of the neuron-specific enolase (NSE) promoter. In multiple lines of NSE-hAPP transgenic mice neuronal overexpression of hAPP was accompanied by an increase in the number of synaptophysin immunoreactive (SYN-IR) presynaptic terminals and in the expression of the growth-associated marker GAP-43. In lines expressing moderate levels of hAPP751 or hAPP695, this effect was more prominent in homozygous than in heterozygous transgenic mice. In contrast, a line with several-fold higher levels of hAPP695 expression showed less increase in SYN-IR presynaptic terminals per amount of hAPP expressed than the lower expressor lines and a decrease in synaptotrophic effects in homozygous compared with heterozygous offspring. Transgenic mice (2-24 months of age) showed no evidence for amyloid deposits or neurodegeneration. These findings suggest that APP may be important for the formation/maintenance of synapses in vivo and that its synaptotrophic effects may be critically dependent on the expression levels of different APP isoforms. Alterations in APP expression, processing or function could contribute to the synaptic pathology seen in AD.

  20. Expression of Human Papillomavirus Type 16 L1 Protein in Transgenic Tobacco Plants

    Institute of Scientific and Technical Information of China (English)

    Hong-Li LIU; Wen-Sheng LI; Ting LEI; Jing ZHENG; Zheng ZHANG; Xiao-Fei YAN; Zhe-Zhi WANG; Yi-Li WANG; Lü-Sheng SI

    2005-01-01

    To develop a plant expression system for the production of the human papillomavirus type 16(HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55 nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV 16L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.

  1. Effect of Homoharringtonine on Bone Morrow CD34+CD7+ Cells in Chronic Granulocytic Leukemia

    Institute of Scientific and Technical Information of China (English)

    LI Yu-feng; DENG Zhi-kui; XUAN Heng-bao; CHEN Bao-an

    2007-01-01

    Objective: To explore the effect of homoharringtonine (HHT) on bone morrow CD34+CD7+ cells in chronic granulocytic leukemia (CGL). Methods: The changes of bone morrow CD34+CD7+ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34+CD7+ cells treated with HHT in vitro were studied. Results: The proportion of CD34+CD7+ cells in CGL (0.145±0.021) was higher than that of normal control (0.052±0.013). The proportion of CD34+CD7+ cells in patients who got cytogenetic responses to HHT (0.072±0.020) decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, (0.137±0.023). the proliferation of CD34+ cells was inhibited and the proportion of CD34+CD7+ cells decreased after cultured with HHT (0.134 in 24 h, 0.126 in 48 h and 0.102 in 72). The apoptosis rate of CD34+CD7+ cells was higher than that in CD34+CD7- cells (35.39%±4.39% versus 24.57%(4.01%, P<0.05) 72 h after culture with HHT. Conclusion: The proportion of CD34+CD7+ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34+CD7+ cells.

  2. 23. Establishment of two transgenic cells stable expression of human cytochrome P450 2C

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM: To clone the human cytochrome P450 2C9 (CYP2C9) and CYP2C18 cDNA and establish two transgenic CHL cell line stable expressing human CYP2C9 and CYP2C18. METHODS:Extracting total RNA from human liver tissue, the human CYP2C9 and CYP2C18 cDNA was amplified with reverse transcription polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. Two transgenic cell line were established by transfecting the recombinant vectors of pREP9-CYP2C9 and pREP9-CYP2C18 to Chinese hamster lung cell CHL. The enzyme activity of CYP2C9 and CYP2C18 catalyze tolbutamide to 4-hydroxy tolbutamide in S9 protein of the cells were determinated by HPLC. RESULTS: The sequence of the two cDNA segments cloned, which were 1540 bp and 1671 bp in length, were identical to those reported by Romkes et al(GenBank accession number: M61855, M61856, J05326) in coding amino acids. The S9 fraction of the established cell lines can metabolize tolbutamide to 4-hydroxy tolbutamide, the tolbutamide-4-hydroxylase activity was found to be 0.465±0.109 and 0.509±0.052 nmol*min-1*(mg S9 protein)-1 (n=3), but was not detectable in parental CHL cell. CONCLUSION: The cDNA of CYP2C9 and CYP2C18 were successfolly cloned and cell lines of CHL-CYP2C9 and CHL-CYP2C18 which efficiently expressed the protein of CYP2C9 and CYP2C18 were established.

  3. Phenobarbital-mediated tumor promotion in transgenic mice with humanized CAR and PXR.

    Science.gov (United States)

    Braeuning, Albert; Gavrilov, Alina; Brown, Susan; Wolf, C Roland; Henderson, Colin J; Schwarz, Michael

    2014-08-01

    The nuclear receptors CAR (constitutive androstane receptor) and possibly PXR (pregnane X receptor) mediate the hepatic effects of phenobarbital (PB) and similar-acting compounds. Although PB is a potent nongenotoxic tumor promoter in rodent liver, epidemiological data from epilepsy patients treated with phenobarbital do not show a specific role of PB in human liver cancer risk. That points to species differences in the susceptibility to tumor promotion by PB, which might be attributed to divergent functions of the PB receptors CAR and PXR in mice and humans. In the present study, male transgenic mice expressing human CAR and PXR were used to detect possible differences between wild-type (WT) and humanized mice in their response to CAR activation in a tumor initiation/promotion experiment with a single injection of the tumor initiator N-nitrosodiethylamine preceding chronic PB treatment for 10 months. Analysis of liver tumor burden revealed that PB strongly promoted the outgrowth of hepatocellular adenoma driven by activated β-catenin in WT mice, whereas the tumor-promoting effect of PB was much less pronounced in the humanized group. In conclusion, the present findings demonstrate that human CAR and PXR support tumor promotion by PB in mouse liver, but to a significantly lesser extent than the WT murine receptors. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. The human apoE7 and apoE4 transgenic mice models

    Institute of Scientific and Technical Information of China (English)

    SUN; Mingzeng; (

    2001-01-01

    [1]Weisgraber, K. H., Apolipoprotein E: Structure-function relationships, Adv. Pro. Chem., 1994, 45: 249-302.[2]Center, G. F., Paoletti, E. G., Apolipoprotein function in health and disease: Insights from natural mutations, Europ. J. Clin. Invest., 1996, 26: 733-746.[3]Browner, J. D., van Dormal, J. J., Muskiet, A. J., Clinical chemistry of common apolipoproteins, J. Chromat. B, 1996, 678: 623-641.[4]Maeda, H., Nakamura, H., Shozo, K. et al., Identification of human apolipoprotein E variant gene: Apolipoprotein E7 (Glu244, 245 Lys244, 245), J. Biochm., 1989, 105: 51-54.[5]Taylor, J. M., Downstream regulatory elements stimulate expression of the human plipoprotein E gene in the liver and suppress expression in the kidney of transgenic mice, Transassoc. Am. Physicians, 1990, 103: 119-128.[6]Smith, J. D., Plump, A. S., Breslow, J. L. et al., Accumulation of human apolipoprotein E in the plasma of transgenic mice, J. Biol. Chem., 1990, 265(25): 14709-14712.[7]Tazio, S., Lee, Y. L., Ji, Z. S. et al., Type III hyperlipoproteinemic phenotype in transgenic mice expressing dysfunctional apolipoprotein E, J. Clin. Invest., 1993, 92(3): 1497-1503.[8]Arn, M. J., Maagdenberg, M., Hofker, M. N. et al., Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia, The J. Biol. Chem., 1993, 268(14): 10540-10545.[9]Qi, Z. H., Ru, K., Sun, M. Z. et al., The gene expression of pME in mouse NIH/3T3 cells and construction of the h-apoE transgenic mice, Chinese Biochem. J., 1997, 13(1): 24-28.[10]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning, A Laboratory Manual, New York: CSH Press, 1989, 81.[11]Hogan, B. L., Manipulation the mouse embryo, A Laboratory Manual, New York: CSH Press, 1989, 89.[12]Wandell, M. R., Rall, S. C., Brennan, S. Jr et al., Apolipoprotein E2-Dunedin (228 Arg-Cys): Anapolipoprotein E2 variant with normal receptor-binding activity, J. Lip. Res., 1990, 31: 534-543.[13

  5. Both lipolysis and hepatic uptake of VLDL are impaired in transgenic mice coexpressing human apolipoprotein E*3Leiden and human apolipoprotein C1

    NARCIS (Netherlands)

    Jong, M.C.; Dahlmans, V.E.H.; Gorp, P.J.J. van; Breuer, M.L.; Mol, M.J.T.M.; Zee, A. van der; Frants, R.R.; Hofker, M.H.; Havekes, L.M.

    1996-01-01

    Transgenic mice overexpressing human APOE*3Leiden are highly susceptible to diet-induced hyperlipoproteinemia and atherosclerosis due to a defect in hepatic uptake of remnant lipoproteins. In addition to the human APOE*3Leiden gene, these mice carry the human APOC1 gene (APOE*3Leiden- C1). To

  6. Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

    Science.gov (United States)

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

    2015-11-01

    With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

  7. SP-LL-37, human antimicrobial peptide, enhances disease resistance in transgenic rice

    Science.gov (United States)

    Cho, Yong Gu; Nou, Ill Sup; Huq, Md. Amdadul; Nogoy, Franz Marielle; Kang, Kwon-Kyoo

    2017-01-01

    Human LL-37 is a multifunctional antimicrobial peptide of cathelicidin family. It has been shown in recent studies that it can serve as a host’s defense against influenza A virus. We now demonstrate in this study how signal peptide LL-37 (SP-LL-37) can be used in rice resistance against bacterial leaf blight and blast. We synthesized LL-37 peptide and subcloned in a recombinant pPZP vector with pGD1 as promoter. SP-LL-37 was introduced into rice plants by Agrobacterium mediated transformation. Stable expression of SP-LL-37 in transgenic rice plants was confirmed by RT-PCR and ELISA analyses. Subcellular localization of SP-LL-37-GFP fusion protein showed evidently in intercellular space. Our data on testing for resistance to bacterial leaf blight and blast revealed that the transgenic lines are highly resistant compared to its wildtype. Our results suggest that LL-37 can be further explored to improve wide-spectrum resistance to biotic stress in rice. PMID:28282452

  8. Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk.

    Science.gov (United States)

    Maga, Elizabeth A; Cullor, James S; Smith, Wayne; Anderson, Gary B; Murray, James D

    2006-01-01

    The addition of human milk components with intrinsic antimicrobial activity to livestock milk by genetic engineering has the potential to benefit milk safety and production as well as the health of the lactating animal. As a model for the dairy cow, we generated transgenic goats that expressed human lysozyme in their milk at 68% of the levels found in human milk. Milk from these transgenic animals had a bacteriostatic effect on both in vitro and in vivo growth of several microorganisms important to the dairy industry. In vitro, milk from transgenic animals was capable of slowing the growth of mastitis-causing strains of Escherichia coli (P transgenic animals. In vivo, milk from transgenic animals supported less bacterial growth than control milk. This transgenic model demonstrates the possibilities offered by genetic engineering to enhance the antimicrobial nature of milk and the udder.

  9. Human apolipoprotein B transgenic SHR/NDmcr-cp rats show exacerbated kidney dysfunction

    Science.gov (United States)

    ASAHINA, Makoto; SHIMIZU, Fumi; OHTA, Masayuki; TAKEYAMA, Michiyasu; TOZAWA, Ryuichi

    2015-01-01

    Nephropathy frequently co-occurs with metabolic syndrome in humans. Metabolic syndrome is a cluster of metabolic diseases including obesity, diabetes, hypertension, and dyslipidemia, and some previous studies revealed that dyslipidemia contributes to the progression of kidney dysfunction. To establish a new nephropathy model with metabolic syndrome, we produced human apolipoprotein B (apoB) transgenic (Tg.) SHR/NDmcr-cp (SHR-cp/cp) rats, in which dyslipidemia is exacerbated more than in an established metabolic syndrome model, SHR-cp/cp rats. Human apoB Tg. SHR-cp/cp rats showed obesity, hyperinsulinemia, hypertension, and severe hyperlipidemia. They also exhibited exacerbated early-onset proteinuria, accompanied by increased kidney injury and increased oxidative and inflammatory markers. Histological analyses revealed the characteristic features of human apoB Tg. SHR-cp/cp rats including prominent glomerulosclerosis with lipid accumulation. Our newly established human apoB Tg. SHR-cp/cp rat could be a useful model for the nephropathy in metabolic syndrome and for understanding the interaction between dyslipidemia and renal dysfunction in metabolic syndrome. PMID:25912321

  10. Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster.

    Science.gov (United States)

    Namciu, Stephanie J; Fournier, R E K

    2004-12-01

    Human matrix attachment regions (MARs) can insulate transgene expression from chromosomal position effects in Drosophila melanogaster. To gain insight into the mechanism(s) by which chromosomal insulation occurs, we studied the expression phenotypes of Drosophila transformants expressing mini-white transgenes in which MAR sequences from the human apoB gene were arranged in a variety of ways. In agreement with previous reports, we found that a single copy of the insulating element was not sufficient for position-independent transgene expression; rather, two copies were required. However, the arrangement of the two elements within the transgene was unimportant, since chromosomal insulation was equally apparent when both copies of the insulator were upstream of the mini-white reporter as when the transcription unit was flanked by insulator elements. Moreover, experiments in which apoB 3' MAR sequences were removed from integrated transgenes in vivo by site-specific recombination demonstrated that MAR sequences were required for the establishment but not for the maintenance of chromosomal insulation. These observations are not compatible with the chromosomal loop model in its simplest form. Alternate mechanisms for MAR function in this system are proposed.

  11. Expression of Human Hepatitis B Virus Surface Antigen Gene in Transgenic Tobacco

    Institute of Scientific and Technical Information of China (English)

    刘玉乐; 王晋芳; 邱并生; 赵淑珍; 田波

    1994-01-01

    Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time. The recombinant plasmid pRoKⅡ-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKⅡ and then introduced into Agrobacterium tumefaciens LBA4404. The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system. It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA. The spherical particles of ψ 22 nm in the leaf extract of trangenic tobacco were observed by immunosorbent electron microscopy.

  12. Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encoding human erythropoietin (hEPO) and governed by regulatory sequences of mouse whey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%. hEPO was expressed in the milk of 16 mice of 39 mice measured by hEPO ELISA kit .The expression level gets over 15 m g/mL.

  13. Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system.

    Science.gov (United States)

    Sumiyoshi, Teiko; Holt, Nathalia G; Hollis, Roger P; Ge, Shundi; Cannon, Paula M; Crooks, Gay M; Kohn, Donald B

    2009-12-01

    Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell

  14. Transient Expression of Transgenic IL-12 in Mouse Liver Triggers Unremitting Inflammation Mimicking Human Autoimmune Hepatitis.

    Science.gov (United States)

    Gil-Farina, Irene; Di Scala, Marianna; Salido, Eduardo; López-Franco, Esperanza; Rodríguez-García, Estefania; Blasi, Mercedes; Merino, Juana; Aldabe, Rafael; Prieto, Jesús; Gonzalez-Aseguinolaza, Gloria

    2016-09-15

    The etiopathogenesis of autoimmune hepatitis (AIH) remains poorly understood. In this study, we sought to develop an animal model of human AIH to gain insight into the immunological mechanisms driving this condition. C57BL/6 mice were i.v. injected with adeno-associated viral vectors encoding murine IL-12 or luciferase under the control of a liver-specific promoter. Organ histology, response to immunosuppressive therapy, and biochemical and immunological parameters, including Ag-specific humoral and cellular response, were analyzed. Mechanistic studies were carried out using genetically modified mice and depletion of lymphocyte subpopulations. Adeno-associated virus IL-12-treated mice developed histological, biochemical, and immunological changes resembling type 1 AIH, including marked and persistent liver mononuclear cell infiltration, hepatic fibrosis, hypergammaglobulinemia, anti-nuclear and anti-smooth muscle actin Abs, and disease remission with immunosuppressive drugs. Interestingly, transgenic IL-12 was short-lived, but endogenous IL-12 expression was induced, and both IL-12 and IFN-γ remained elevated during the entire study period. IFN-γ was identified as an essential mediator of liver damage, and CD4 and CD8 T cells but not NK, NKT, or B cells were essential executors of hepatic injury. Furthermore, both MHC class I and MHC class II expression was upregulated at the hepatocellular membrane, and induction of autoreactive liver-specific T cells was detected. Remarkably, although immunoregulatory mechanisms were activated, they only partially mitigated liver damage. Thus, low and transient expression of transgenic IL-12 in hepatocytes causes loss of tolerance to hepatocellular Ags, leading to chronic hepatitis resembling human AIH type 1. This model provides a practical tool to explore AIH pathogenesis and novel therapies.

  15. Conditional E2F1 activation in transgenic mice causes testicular atrophy and dysplasia mimicking human CIS

    DEFF Research Database (Denmark)

    Agger, Karl; Santoni-Rugiu, Eric; Holmberg, Christian;

    2005-01-01

    E2F1 is a crucial downstream effector of the retinoblastoma protein (pRB) pathway. To address the consequences of short-term increase in E2F1 activity in adult tissues, we generated transgenic mice expressing the human E2F1 protein fused to the oestrogen receptor (ER) ligand-binding domain...

  16. Handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins in transgenic mice

    DEFF Research Database (Denmark)

    Kragh, Peter M; Pedersen, Christina B; Schmidt, Stine P;

    2007-01-01

    Abstract To investigate the in vivo handling of human short-chain acyl-CoA dehydrogenase (SCAD) variant proteins, three transgenic mouse lines were produced by pronuclear injection of cDNA encoding the wild-type, hSCAD-wt, and two disease causing folding variants hSCAD-319C > T and hSCAD-625G > A...

  17. Renal tubule-specific expression and urinary secretion of human growth hormone: a kidney-based transgenic bioreactor growth.

    Science.gov (United States)

    Zhu, Xinhua; Cheng, Jin; Huang, Liwei; Gao, Jin; Zhang, Zhong-Ting; Pak, Joanne; Wu, Xue-Ru

    2003-04-01

    Tissue-specific expression of human genes and secretion of human proteins into the body fluids in transgenic animals provides an important means of manufacturing large-quantity and high-quality pharmaceuticals. The present study demonstrates using transgenic mice that a 3.0 kb promoter of the mouse Tamm-Horsfall protein (THP, or uromodulin) gene directs the specific expression of human growth hormone (hGH) gene in the kidney followed by the secretion of hGH protein into the urine. hGH expression was detected in renal tubules that actively produce the THP, that is, the ascending limb of Henle's loop and distal convoluted tubules. Up to 500 ng/ml of hGH was detected in the urine, and this level remained constant throughout the 10-month observation period. hGH was also detectable in the stomach epithelium and serum in two of the transgenic lines, suggesting position-dependent effects of the transgene and leakage of hGH from the site of synthesis into the bloodstream, respectively. These results indicate that the 3.0 kb mouse THP promoter is primarily kidney-specific and can be used to convert kidney into a bioreactor in transgenic animals to produce recombinant proteins. Given the capacity of urine production independent of age, sex and lactation, the ease of urinary protein purification, and the potentially distinct machinery for post-translational modifications in the kidney epithelial cells, the kidney-based transgenic bioreactor may offer unique opportunities for producing certain complex pharmaceuticals.

  18. Plasma clearance of human low-density lipoprotein in human apolipoprotein B transgenic mice is related to particle diameter.

    Science.gov (United States)

    Berneis, Kaspar; Shames, David M; Blanche, Patricia J; La Belle, Michael; Rizzo, Manfredi; Krauss, Ronald M

    2004-04-01

    To test for intrinsic differences in metabolic properties of low-density lipoprotein (LDL) as a function of particle size, we examined the kinetic behavior of 6 human LDL fractions ranging in size from 251 to 265 A injected intravenously into human apolipoprotein (apo) B transgenic mice. A multicompartmental model was formulated and fitted to the data by standard nonlinear regression using the Simulation, Analysis and Modeling (SAAM II) program. Smaller sized LDL particles (251 to 257 A) demonstrated a significantly slower fractional catabolic rate (FCR) (0.050 +/- 0.045 h(-1)) compared with particles of larger size (262 to 265 A) (0.134 +/- -0.015 h(-1), P particles are cleared more slowly from plasma than larger LDL and are exchanged more slowly with the extravascular space. This might be due to compositional or structural features of smaller LDL that lead to retarded clearance.

  19. Generation of transgenic cattle expressing human β-defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection.

    Science.gov (United States)

    Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong

    2016-03-01

    Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection.

  20. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β -Globin Gene with the IVSI-6 Thalassemia Mutation.

    Science.gov (United States)

    Breveglieri, Giulia; Mancini, Irene; Bianchi, Nicoletta; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Rubini, Michele; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto; Finotti, Alessia

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin (mu) α-globin2/(hu) β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia.

  1. Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements.

    Science.gov (United States)

    Samiec, M; Skrzyszowska, M

    2011-01-01

    Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.

  2. Recombinant human proinsulin from transgenic corn endosperm: solvent screening and extraction studies

    Directory of Open Access Journals (Sweden)

    C. S. Farinas

    2007-09-01

    Full Text Available Recombinant pharmaceutical proteins are being produced in different systems such as bacteria and mammalian cell cultures. The use of transgenic plants as bioreactors has recently arisen as an alternative system offering many practical and economic advantages. However, finding an optimum strategy for the downstream processing (DSP of recombinant proteins from plants still remains a challenge. In this work, we studied the extraction of recombinant human proinsulin (rhProinsulin produced in the endosperm of transgenic corn seeds. An efficient extraction solvent was selected and the effects of temperature, solvent-to-solid ratio, time, and impeller rotational speed on the extraction were evaluated using an experimental design. After an extraction kinetics study, temperature was further evaluated to maximize rhProinsulin concentration in the extracts and to minimize the native corn components carbohydrates, phenolic compounds, and proteins. A high efficiency condition for extracting rhProinsulin with the selected solvent - 50 mM sodium bicarbonate buffer pH 10.0 and 5 mM DTT - was an extraction time of 2 h at a solvent-to-solid ratio of 10:1 and 25º C. The maximum rhProinsulin concentration in the extracts at that condition was 18.87 mg l-1 or 0.42% of the total soluble protein. These values are within the range in which the production of pharmaceutical proteins in plants can be competitive with other expression systems. The results presented provide information for the development of an additional production platform for the hormone insulin.

  3. Transgenic milk containing recombinant human lactoferrin modulates the intestinal flora in piglets.

    Science.gov (United States)

    Hu, Wenping; Zhao, Jie; Wang, Jianwu; Yu, Tian; Wang, Jing; Li, Ning

    2012-06-01

    Lactoferrin (LF) is a beneficial multifunctional protein in milk. The objective of this study was to determine whether bovine transgenic milk containing recombinant human lactoferrin (rhLF) can modulate intestinal flora in the neonatal pig as an animal model for the human infant. We fed 7-day-old piglets (i) ordinary whole milk (OM), (ii) a 1:1 mixture of OM and rhLF milk (MM), or (iii) rhLF milk (LFM). LFM provided better average daily mass gain than OM (P = 0.007). PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis revealed that the LFM piglets exhibited more diversity of the intestinal flora than the OM group. Except for the colon in the LFM group, an increasing trend in microbial diversity occurred from the duodenum to the colon. Fecal flora was not different across different ages or different treatment groups, but a cluster analysis showed that the fecal flora of OM- and MM-fed piglets had a higher degree of similarity than that of LFM-fed piglets. Based on culture-based bacterial counts of intestinal content samples, concentrations of Salmonella spp. in the colon and of Escherichia coli throughout the intestine were reduced with LFM (P intestine were also increased with LFM (P ≤ 0.01). We suggest that rhLF can modulate the intestinal flora in piglets.

  4. GFAP expression and social deficits in transgenic mice overexpressing human sAPPα

    Science.gov (United States)

    Bailey, Antoinette R; Hou, Huayan; Song, Min; Obregon, Demian F; Portis, Samantha; Barger, Steven; Shytle, Doug; Stock, Saundra; Mori, Takashi; Sanberg, Paul G; Murphy, Tanya; Tan, Jun

    2013-01-01

    Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein-alpha (sAPPα), the product of α-secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL-6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism-like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL-6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild-type mice treated with sAPPα, displayed suppressed β-tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment. PMID:23840007

  5. mRNA Expression of Vimentin Gene in Lens of Transgenic Mouse and DNA Amplification in Human Cataracts

    Institute of Scientific and Technical Information of China (English)

    YanLi; XienpingLiu; 等

    1995-01-01

    Purpose:To investigate the role of vimentin gene in cataractogenesis.Methods:The12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs.841injected embryos were transferred into oviducts of pseudopregnant recipient females.of which 12pregnant mice gave birth to 49offsping mice.The integration and expression of exogenous gene in the offsping were analysed by Southern and Northern blot byhridizations,In the human senile cataract,the lens vimentin gene was analyzed with the chicken vi-mentin gene probe.Results:It showed that four of F1offspring were transgenic mice in which the chicken vimenttin gene was integrated in their genomes.The transgenic band was12kb,similar to the12.7kb chicken vimentin fragment injected.One2kbvi-mentin mRNAwas visualized on E2 mouse lens blot.which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse.In the humansenile cataract lens,12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot anal-ysis,It implies that the Formation of human senile cataract may be associated with the amplification of vimentin gene.Conclusions:We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study.It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice.The cause of the gene ampli-fication in human ctaract remains for further investigation.Eye Science 1995;11:113-116.

  6. Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer.

    Science.gov (United States)

    Wilken, Lisa R; Nikolov, Zivko L

    2010-01-01

    Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.

  7. Cytoprotective effect of recombinant human erythropoietin produced in transgenic tobacco plants.

    Directory of Open Access Journals (Sweden)

    Farooqahmed S Kittur

    Full Text Available Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P (20 U/ml provides 2-fold better cytoprotection (44% to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M (21%. The cytoprotective effect of the asialo-rhuEPO(P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2 and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.

  8. The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice.

    Science.gov (United States)

    Gurskiy, Yaroslav G; Garbuz, David G; Soshnikova, Nataliya V; Krasnov, Aleksey N; Deikin, Alexei; Lazarev, Vladimir F; Sverchinskyi, Dmitry; Margulis, Boris A; Zatsepina, Olga G; Karpov, Vadim L; Belzhelarskaya, Svetlana N; Feoktistova, Evgenia; Georgieva, Sofia G; Evgen'ev, Michael B

    2016-11-01

    The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.

  9. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Science.gov (United States)

    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  10. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    Directory of Open Access Journals (Sweden)

    Jianhua Gong

    Full Text Available Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency, hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  11. Beta-catenin accelerates human papilloma virus type-16 mediated cervical carcinogenesis in transgenic mice.

    Directory of Open Access Journals (Sweden)

    Gülay Bulut

    Full Text Available Human papilloma virus (HPV is the principal etiological agent of cervical cancer in women, and its DNA is present in virtually all of these tumors. However, exposure to the high-risk HPV types alone is insufficient for tumor development. Identifying specific collaborating factors that will lead to cervical cancer remains an unanswered question, especially because millions of women are exposed to HPV. Our earlier work using an in vitro model indicated that activation of the canonical Wnt pathway in HPV-positive epithelial cells was sufficient to induce anchorage independent growth. We therefore hypothesized that constitutive activation of this pathway might function as the "second hit." To address this possibility, we developed two double-transgenic (DT mouse models, K14-E7/ΔN87βcat and K14-HPV16/ΔN87βcat that express either the proteins encoded by the E7 oncogene or the HPV16 early region along with constitutively active β-catenin, which was expressed by linking it to the keratin-14 (K14 promoter. We initiated tumor formation by treating all groups with estrogen for six months. Invasive cervical cancer was observed in 11% of the K14-ΔN87βcat mice, expressing activated β-catenin and in 50% of the animals expressing the HPV16 E7 oncogene. In double-transgenic mice, coexpression of β-catenin and HPV16 E7 induced invasive cervical cancer at about 7 months in 94% of the cases. We did not observe cervical cancer in any group unless the mice were treated with estrogen. In the second model, K14-HPV16 mice suffered cervical dysplasias, but this phenotype was not augmented in HPV16/ΔN87βcat mice. In summary, the phenotypes of the K14-E7/ΔN87βcat mice support the hypothesis that activation of the Wnt/β-catenin pathway in HPV-associated premalignant lesions plays a functional role in accelerating cervical carcinogenesis.

  12. High-level, erythroid specific, expression of the human α-globin gene in transgenic mice and the production of human haemoglobin in murine erythrocytes.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); M. Vidal; J. Kaeda; L. Luzzatto; D.R. Greaves (David); F.G. Grosveld (Frank)

    1989-01-01

    textabstractUsing the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of

  13. Improvements in human health through production of human milk proteins in transgenic food plants.

    Science.gov (United States)

    Arakawa, T; Chong, D K; Slattery, C W; Langridge, W H

    1999-01-01

    Plants are particularly suitable bioreactors for the production of proteins, as their eukaryotic nature frequently directs the appropriate post-translational modifications of recombinant proteins to retain native biological activity. The autotrophic growth of plants makes this in vivo biosynthesis system economically competitive for supplementation or replacement of conventional production systems in the future. For the production of biologically active proteins, food plants provide the advantage of direct delivery via consumption of transformed plant tissues. Here we describe the production of recombinant human milk proteins in food plants for improvements in human nutrition and health, with emphasis on enhanced nutrition for non-breast fed infants as well as children and adults. Nutritional improvements in edible plants generated through advancements in recombinant DNA technology are rapidly repositioning the world for enjoyment of a more healthful diet for humans in all age groups.

  14. Production of multiple transgenic Yucatan miniature pigs expressing human complement regulatory factors, human CD55, CD59, and H-transferase genes.

    Directory of Open Access Journals (Sweden)

    Young-Hee Jeong

    Full Text Available The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC compared with the human umbilical vein endothelial cells (HUVEC. Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative

  15. Transgenic mice with -6A haplotype of the human angiotensinogen gene have increased blood pressure compared with -6G haplotype.

    Science.gov (United States)

    Jain, Sudhir; Tillinger, Andrej; Mopidevi, Brahmaraju; Pandey, Varunkumar G; Chauhan, Chetankumar K; Fiering, Steven N; Warming, Soren; Kumar, Ashok

    2010-12-24

    Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at -6, and the -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with -6G. We have found that the hAGT gene has three additional SNPs (A/G at -1670, C/G at -1562, and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A, and variants -1670G, -1562G, and -1561G almost always occur with -6G. Therefore, the hAGT gene may be subdivided into either -6A or -6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with -6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the -6A or -6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared with -6G haplotype. Our studies explain the molecular mechanism involved in association of the -6A allele of the hAGT gene with hypertension.

  16. [Transgenic animals bioreactors].

    Science.gov (United States)

    Gou, Ke-Mian; An, Xiao-Rong; Tian, Jian-Hui; Chen, Yong-Fu

    2002-01-01

    The production of human recombinant proteins in milk of transgenic farm animals offers a safe, very cost-effective source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods. This review has summarized the current status of gene selection, vector construct, transgenic methods, economics, and obvious potential in transgenic animals bioreactors. Recently, a more powerful approach was adopted in the transgenic animals founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic farm animals, product consistency, and time of product development. The successful adaptation of Cre-/lox P-mediated site-specific DNA recombination systems in farm animals will offer unprecedented possibilities for generating transgenic animals.

  17. Dietary cholesterol fails to stimulate the human cholesterol 7alpha-hydroxylase gene (CYP7A1) in transgenic mice.

    Science.gov (United States)

    Agellon, Luis B; Drover, Victor A B; Cheema, Sukhinder K; Gbaguidi, G Franck; Walsh, Annemarie

    2002-06-07

    Dietary cholesterol has been shown to have a stimulatory effect on the murine cholesterol 7alpha-hydroxylase gene (Cyp7a1), but its effect on human cholesterol 7alpha-hydroxylase gene (CYP7A1) expression in vivo is not known. A transgenic mouse strain harboring the human CYP7A1 gene and homozygous for the disrupted murine Cyp7a1 gene was created. Cholesterol feeding increased the expression of the endogenous modified Cyp7a1 allele but failed to stimulate the human CYP7A1 transgene. In transfected hepatoma cells, 25-hydroxycholesterol increased murine Cyp7a1 gene promoter activity, whereas the human CYP7A1 gene promoter was unresponsive. Electrophoretic mobility shift assays demonstrated the interaction of the liver X receptor alpha (LXRalpha): retinoid X receptor (RXR) heterodimer, a transcription factor complex that is activated by oxysterols, with the murine Cyp7a1 gene promoter, whereas no binding to the human CYP7A1 gene promoter was detected. The results demonstrate that the human CYP7A1 gene is not stimulated by dietary cholesterol in the intact animal, and this is attributable to the inability of the CYP7A1 gene promoter to interact with LXRalpha:RXR.

  18. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation

    Science.gov (United States)

    Lavitrano, Marialuisa; Bacci, Maria Laura; Forni, Monica; Lazzereschi, Davide; Di Stefano, Carla; Fioretti, Daniela; Giancotti, Paola; Marfé, Gabriella; Pucci, Loredana; Renzi, Luigina; Wang, Hongjun; Stoppacciaro, Antonella; Stassi, Giorgio; Sargiacomo, Massimo; Sinibaldi, Paola; Turchi, Valeria; Giovannoni, Roberto; Della Casa, Giacinto; Seren, Eraldo; Rossi, Giancarlo

    2002-01-01

    A large number of hDAF transgenic pigs to be used for xenotransplantation research were generated by using sperm-mediated gene transfer (SMGT). The efficiency of transgenesis obtained with SMGT was much greater than with any other method. In the experiments reported, up to 80% of pigs had the transgene integrated into the genome. Most of the pigs carrying the hDAF gene transcribed it in a stable manner (64%). The great majority of pigs that transcribed the gene expressed the protein (83%). The hDAF gene was transmitted to progeny. Expression was stable and found in caveolae as it is in human cells. The expressed gene was functional based on in vitro experiments performed on peripheral blood mononuclear cells. These results show that our SMGT approach to transgenesis provides an efficient procedure for studies involving large animal models. PMID:12393815

  19. Use of human lysozyme transgenic goat milk in cheese making: effects on lactic acid bacteria performance.

    Science.gov (United States)

    Scharfen, E C; Mills, D A; Maga, E A

    2007-09-01

    Genetically engineered goats expressing elevated levels of the antimicrobial enzyme lysozyme in their milk were developed to improve udder health, product shelf life, and consumer well-being. The purpose of this study was to evaluate the effect of lysozyme on the development of lactic acid bacteria (LAB) throughout the cheese-making process. Raw and pasteurized milk from 7 lysozyme transgenic goats and 7 breed-, age-, and parity-matched nontransgenic controls was transformed into cheeses by using industry methods, and their microbiological load was evaluated. The numbers of colony-forming units of LAB were determined for raw and pasteurized goat milk, whey, and curd at d 2 and at d 6 or 7 of production. Selective plating media were used to enumerate lactococcal species separately from total LAB. Although differences in the mean number of colony-forming units between transgenic and control samples in raw milk, whey, and cheese curd were non-significant for both total LAB and lactococcal species from d 2 of production, a significant decrease was observed in both types of LAB among d 6 transgenic raw milk cheese samples. In pasteurized milk trials, a significant decrease in LAB was observed only in the raw milk of transgenic animals. These results indicate that lysozyme transgenic goat milk is not detrimental to LAB growth during the cheese-making process.

  20. Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice.

    Science.gov (United States)

    Padilla, Danielle; Béringue, Vincent; Espinosa, Juan Carlos; Andreoletti, Olivier; Jaumain, Emilie; Reine, Fabienne; Herzog, Laetitia; Gutierrez-Adan, Alfonso; Pintado, Belen; Laude, Hubert; Torres, Juan Maria

    2011-03-01

    A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.

  1. Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice.

    Directory of Open Access Journals (Sweden)

    Danielle Padilla

    2011-03-01

    Full Text Available A new variant of Creutzfeldt Jacob Disease (vCJD was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met allele of human PrP at codon 129 (tg650 and tg340 with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.

  2. Transgenic expression of human heme oxygenase-1 in pigs confers resistance against xenograft rejection during ex vivo perfusion of porcine kidneys.

    Science.gov (United States)

    Petersen, Björn; Ramackers, Wolf; Lucas-Hahn, Andrea; Lemme, Erika; Hassel, Petra; Queisser, Anna-Lisa; Herrmann, Doris; Barg-Kues, Brigitte; Carnwath, Joseph W; Klose, Johannes; Tiede, Andreas; Friedrich, Lars; Baars, Wiebke; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

    2011-01-01

    The major immunological hurdle to successful porcine-to-human xenotransplantation is the acute vascular rejection (AVR), characterized by endothelial cell (EC) activation and perturbation of coagulation. Heme oxygenase-1 (HO-1) and its derivatives have anti-apoptotic, anti-inflammatory effects and protect against reactive oxygen species, rendering HO-1 a promising molecule to control AVR. Here, we report the production and characterization of pigs transgenic for human heme oxygenase-1 (hHO-1) and demonstrate significant protection in porcine kidneys against xenograft rejection in ex vivo perfusion with human blood and transgenic porcine aortic endothelial cells (PAEC) in a TNF-α-mediated apoptosis assay. Transgenic and non-transgenic PAEC were tested in a TNF-α-mediated apoptosis assay. Expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin) was measured by real-time PCR. hHO-1 transgenic porcine kidneys were perfused with pooled and diluted human AB blood in an ex vivo perfusion circuit. MHC class-II up-regulation after induction with IFN-γ was compared between wild-type and hHO-1 transgenic PAEC. Cloned hHO-1 transgenic pigs expressed hHO-1 in heart, kidney, liver, and in cultured ECs and fibroblasts. hHO-1 transgenic PAEC were protected against TNF-α-mediated apoptosis. Real-time PCR revealed reduced expression of adhesion molecules like ICAM-1, VCAM-1, and E-selectin. These effects could be abrogated by the incubation of transgenic PAECs with the specific HO-1 inhibitor zinc protoporphorine IX (Zn(II)PPIX, 20 μm). IFN-γ induced up-regulation of MHC class-II molecules was significantly reduced in PAECs from hHO-1 transgenic pigs. hHO-1 transgenic porcine kidneys could successfully be perfused with diluted human AB-pooled blood for a maximum of 240 min (with and without C1 inh), while in wild-type kidneys, blood flow ceased after ∼60 min. Elevated levels of d-Dimer and TAT were detected, but no significant consumption of fibrinogen and

  3. Expression of human hormone-sensitive lipase in white adipose tissue of transgenic mice increases lipase activity but does not enhance in vitro lipolysis.

    Science.gov (United States)

    Lucas, Stéphanie; Tavernier, Geneviève; Tiraby, Claire; Mairal, Aline; Langin, Dominique

    2003-01-01

    Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters (CEs). The enzyme is highly expressed in adipose tissues (ATs), where it is thought to play an important role in fat mobilization. The purpose of the present work was to study the effect of a physiological increase of HSL expression in vivo. Transgenic mice were produced with a 21 kb human genomic fragment encompassing the exons encoding the adipocyte form of HSL. hHSL mRNA was expressed at 3-fold higher levels than murine HSL mRNA in white adipocytes. Transgene expression was also observed in brown adipose tissue (BAT) and skeletal muscle. The human protein was detected in ATs of transgenic (Tg) mice. The hydrolytic activities against triacylglycerol (TG), diacylglycerol (DG) analog, and CE were increased in transgenic mouse AT. However, cAMP-inducible adipocyte lipolysis was lower in transgenic animals. In the B6CBA genetic background, transgenic mice up to 14 weeks of age showed lower body weight and fat mass. The phenotype was not observed in older animals and in mice fed a high-fat diet (HFD). In the OF1 genetic background, there was no difference in fat mass of mice fed ad libitum. However, transgenic mice became leaner than their wild-type (WT) littermates after a 4 day calorie restriction. The data show that overexpression of HSL, despite increased lipase activity, does not lead to enhanced lipolysis.

  4. Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

    Science.gov (United States)

    Kishino, Yoshikazu; Seki, Tomohisa; Fujita, Jun; Yuasa, Shinsuke; Tohyama, Shugo; Kunitomi, Akira; Tabei, Ryota; Nakajima, Kazuaki; Okada, Marina; Hirano, Akinori; Kanazawa, Hideaki; Fukuda, Keiichi

    2014-01-01

    Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

  5. Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

    NARCIS (Netherlands)

    Meng, L.; Wan, Y.; Sun, Y.; Zhang, Y.; Wang, Z.; Song, Y.; Wang, F.

    2013-01-01

    Background - Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos.

  6. Preparation and Amplification of Colony of Goat Transgenic Fetal Fibroblast and Mammary Gland Epithelial Cell with Human Lactoferrin Gene

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-ling; LIU Feng-jun; ZHANG Yong

    2009-01-01

    [Objective] The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%, 50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture, neo gene was as screened gene, genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium, 100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%; MGE: 33.33% vs. 6.67%). Compared with control, con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%), confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell, inserting and diagnosing ideal vector, and can save expense and time for transgenic animal production.

  7. Crohn's disease adherent-invasive Escherichia coli colonize and induce strong gut inflammation in transgenic mice expressing human CEACAM.

    Science.gov (United States)

    Carvalho, Frédéric A; Barnich, Nicolas; Sivignon, Adeline; Darcha, Claude; Chan, Carlos H F; Stanners, Clifford P; Darfeuille-Michaud, Arlette

    2009-09-28

    Abnormal expression of CEACAM6 is observed at the apical surface of the ileal epithelium in Crohn's disease (CD) patients, and CD ileal lesions are colonized by pathogenic adherent-invasive Escherichia coli (AIEC). We investigated the ability of AIEC reference strain LF82 to colonize the intestinal mucosa and to induce inflammation in CEABAC10 transgenic mice expressing human CEACAMs. AIEC LF82 virulent bacteria, but not nonpathogenic E. coli K-12, were able to persist in the gut of CEABAC10 transgenic mice and to induce severe colitis with reduced survival rate, marked weight loss, increased rectal bleeding, presence of erosive lesions, mucosal inflammation, and increased proinflammatory cytokine expression. The colitis depended on type 1 pili expression by AIEC bacteria and on intestinal CEACAM expression because no sign of colitis was observed in transgenic mice infected with type 1 pili-negative LF82-Delta fimH isogenic mutant or in wild-type mice infected with AIEC LF82 bacteria. These findings strongly support the hypothesis that in CD patients having an abnormal intestinal expression of CEACAM6, AIEC bacteria via type 1 pili expression can colonize the intestinal mucosa and induce gut inflammation. Thus, targeting AIEC adhesion to gut mucosa represents a new strategy for clinicians to prevent and/or to treat ileal CD.

  8. Transgenic Primate Research Paves the Path to a Better Animal Model: Are We a Step Closer to Curing Inherited Human Genetic Disorders?

    Institute of Scientific and Technical Information of China (English)

    Anthony W.S. Chan

    2009-01-01

    While the advancement of transgenic primate models has led to a new era in modeling human conditions and has a clear impact on elucidating the mechanism of human genetic diseases, some thoughts should be considered if non-human primates are the appropriate model.

  9. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  10. Myeloid Engraftment in Humanized Mice: Impact of Granulocyte-Colony Stimulating Factor Treatment and Transgenic Mouse Strain.

    Science.gov (United States)

    Coughlan, Alice M; Harmon, Cathal; Whelan, Sarah; O'Brien, Eóin C; O'Reilly, Vincent P; Crotty, Paul; Kelly, Pamela; Ryan, Michelle; Hickey, Fionnuala B; O'Farrelly, Cliona; Little, Mark A

    2016-04-01

    Poor myeloid engraftment remains a barrier to experimental use of humanized mice. Focusing primarily on peripheral blood cells, we compared the engraftment profile of NOD-scid-IL2Rγc(-/-) (NSG) mice with that of NSG mice transgenic for human membrane stem cell factor (hu-mSCF mice), NSG mice transgenic for human interleukin (IL)-3, granulocyte-macrophage-colony stimulating factor (GM-CSF), and stem cell factor (SGM3 mice). hu-mSCF and SGM3 mice showed enhanced engraftment of human leukocytes compared to NSG mice, and this was reflected in the number of human neutrophils and monocytes present in these strains. Importantly, discrete classical, intermediate, and nonclassical monocyte populations were identifiable in the blood of NSG and hu-mSCF mice, while the nonclassical population was absent in the blood of SGM3 mice. Granulocyte-colony stimulating factor (GCSF) treatment increased the number of blood monocytes in NSG and hu-mSCF mice, and neutrophils in NSG and SGM3 mice; however, this effect appeared to be at least partially dependent on the stem cell donor used to engraft the mice. Furthermore, GCSF treatment resulted in a preferential expansion of nonclassical monocytes in both NSG and hu-mSCF mice. Human tubulointerstitial CD11c(+) cells were present in the kidneys of hu-mSCF mice, while monocytes and neutrophils were identified in the liver of all strains. Bone marrow-derived macrophages prepared from NSG mice were most effective at phagocytosing polystyrene beads. In conclusion, hu-mSCF mice provide the best environment for the generation of human myeloid cells, with GCSF treatment further enhancing peripheral blood human monocyte cell numbers in this strain.

  11. A haplotype of human angiotensinogen gene containing -217A increases blood pressure in transgenic mice compared with -217G.

    Science.gov (United States)

    Jain, Sudhir; Vinukonda, Govindaiah; Fiering, Steven N; Kumar, Ashok

    2008-12-01

    The human angiotensinogen (hAGT) gene contains an A/G polymorphism at -217, and frequency of -217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant -217A almost always occurs with -532T, -793A, and -1074T, whereas variant -217G almost always occurs with -532C, -793G, and -1074G. Since allele -6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, -6A:-217A (AA) and -6A:-217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by approximately 40%, and plasma angiotensin II level is increased by approximately 50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure.

  12. Effects of Metformin on Tissue Oxidative and Dicarbonyl Stress in Transgenic Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein.

    Science.gov (United States)

    Malínská, Hana; Oliyarnyk, Olena; Škop, Vojtěch; Šilhavý, Jan; Landa, Vladimír; Zídek, Václav; Mlejnek, Petr; Šimáková, Miroslava; Strnad, Hynek; Kazdová, Ludmila; Pravenec, Michal

    2016-01-01

    Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its "pleiotropic" effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action-studied by gene expression profiling in the liver-revealed deregulated genes from inflammatory and insulin signaling, AMP

  13. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

    Science.gov (United States)

    Zeng, Fang; Li, Zicong; Zhu, Qingchun; Dong, Rui; Zhao, Chengcheng; Li, Guoling; Li, Guo; Gao, Wenchao; Jiang, Gelong; Zheng, Enqin; Cai, Gengyuan; Moisyadi, Stefan; Urschitz, Johann; Yang, Huaqiang; Liu, Dewu; Wu, Zhenfang

    2017-01-01

    The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins. PMID:28117418

  14. Human leukocyte antigen class II transgenic mouse model unmasks the significant extrahepatic pathology in toxic shock syndrome.

    Science.gov (United States)

    Tilahun, Ashenafi Y; Marietta, Eric V; Wu, Tsung-Teh; Patel, Robin; David, Chella S; Rajagopalan, Govindarajan

    2011-06-01

    Among the exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes, the superantigens (SAgs) are the most potent T-cell activators known to date. SAgs are implicated in several serious diseases including toxic shock syndrome (TSS), Kawasaki disease, and sepsis. However, the immunopathogenesis of TSS and other diseases involving SAgs are still not completely understood. The commonly used conventional laboratory mouse strains do not respond robustly to SAgs in vivo. Therefore, they must be artificially rendered susceptible to TSS by using sensitizing agents such as d-galactosamine (d-galN), which skews the disease exclusively to the liver and, hence, is not representative of the disease in humans. SAg-induced TSS was characterized using transgenic mice expressing HLA class II molecules that are extremely susceptible to TSS without d-galN. HLA-DR3 transgenic mice recapitulated TSS in humans with extensive multiple-organ inflammation affecting the lung, liver, kidneys, heart, and small intestines. Heavy infiltration with T lymphocytes (both CD4(+) and CD8+), neutrophils, and macrophages was noted. In particular, the pathologic changes in the small intestines were extensive and accompanied by significantly altered absorptive functions of the enterocytes. In contrast to massive liver failure alone in the d-galN sensitization model of TSS, findings of the present study suggest that gut dysfunction might be a key pathogenic event that leads to high morbidity and mortality in humans with TSS.

  15. Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors.

    Science.gov (United States)

    Zeng, Fang; Li, Zicong; Zhu, Qingchun; Dong, Rui; Zhao, Chengcheng; Li, Guoling; Li, Guo; Gao, Wenchao; Jiang, Gelong; Zheng, Enqin; Cai, Gengyuan; Moisyadi, Stefan; Urschitz, Johann; Yang, Huaqiang; Liu, Dewu; Wu, Zhenfang

    2017-01-24

    The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

  16. Production of human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor.

    Science.gov (United States)

    McDonald, Karen A; Hong, Lo Ming; Trombly, David M; Xie, Qing; Jackman, Alan P

    2005-01-01

    Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two-compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant alpha-1-antitrypsin (rAAT). Expression of rAAT is controlled by the rice alpha-amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactor's relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100-247 mg/L (4-10% of the total extracellular protein) in the cell compartment at 5-6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.

  17. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. (Cincinnati College of Medicine, OH (USA))

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

  18. Establishment and evaluation of a transgenic mouse model of arthritis induced by overexpressing human tumor necrosis factor alpha

    Directory of Open Access Journals (Sweden)

    Ge Li

    2016-04-01

    Full Text Available Tumor necrosis factor alpha (TNFα plays a key role in the pathogenesis of rheumatoid arthritis (RA. Blockade of TNFα by monoclonal antibody has been widely used for the therapy of RA since the 1990s; however, its mechanism of efficacy, and potential safety concerns of the treatment are still not fully understood. This study sought to establish a transgenic arthritic mouse model by overexpressing human TNFα (hTNFα and to apply this model as a means to evaluate therapeutic consequences of TNFα inhibitors. The transgenic mouse line (TgTC with FVB background was generated by incorporating 3′-modified hTNFα gene sequences. A progressively erosive polyarthritis developed in the TgTC mice, with many characteristics observed in human rheumatoid arthritis, including polyarticular swelling, impairment of movement, synovial hyperplasia, and cartilage and bone erosion. Gene expression analysis demonstrated that hTNFα is not only expressed in hyperplastic synovial membrane, but also in tissues without lesions, including brain, lung and kidney. Treatment of the TgTC mice with anti-hTNFα monoclonal antibodies (mAb significantly decreased the level of hTNFα in the diseased joint and effectively prevented development of arthritis in a dose-dependent response fashion. Our results indicated that the TgTC mice represent a genetic model which can be used to comprehensively investigate the pathogenesis and therapeutics of TNFα-related diseases.

  19. Transmission of prion strains in a transgenic mouse model overexpressing human A53T mutated α-synuclein.

    Science.gov (United States)

    Mougenot, Anne-Laure J; Bencsik, Anna; Nicot, Simon; Vulin, Johann; Morignat, Eric; Verchère, Jérémy; Bétemps, Dominique; Lakhdar, Latefa; Legastelois, Stéphane; Baron, Thierry G

    2011-05-01

    There is a growing interest in the potential roles of misfolded protein interactions in neurodegeneration. To investigate this issue, we inoculated 3 prion strains intracerebrally into transgenic (TgM83) mice that overexpress human A53T α-synuclein. In comparison to nontransgenic controls, there was a striking decrease in the incubation periods of scrapie, classic and H-type bovine spongiform encephalopathies(C-BSE and H-BSE), with conservation of the histopathologic and biochemical features characterizing these 3 prion strains. TgM83 mice died of scrapie or C-BSE prion diseases before accumulating the insoluble and phosphorylated forms of α-synuclein specific to late stages of synucleinopathy. In contrast, the median incubation time for TgM83 mice inoculated with H-BSE was comparable to that observed when these mice were uninfected, thereby allowing the development of molecular alterations of α-synuclein. The last 4 mice of this cohort exhibited early accumulations of H-BSE prion protein along with α-synuclein pathology. The results indicate that a prion disease was triggered concomitantly with an overt synucleinopathy in some transgenic mice overexpressing human A53T α-synuclein after intracerebral inoculation with an H-BSE prion strain.

  20. c-RET molecule in malignant melanoma from oncogenic RET-carrying transgenic mice and human cell lines.

    Directory of Open Access Journals (Sweden)

    Yuichiro Ohshima

    Full Text Available Malignant melanoma is one of the most aggressive cancers and its incidence worldwide has been increasing at a greater rate than that of any other cancer. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice spontaneously develop malignant melanoma. In this study, we showed that expression levels of intrinsic c-Ret, glial cell line-derived neurotrophic factor (Gdnf and Gdnf receptor alpha 1 (Gfra1 transcripts in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors. These results suggest that not only introduced oncogenic RET but also intrinsic c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then showed that c-RET and GDNF transcript expression levels in human malignant melanoma cell lines (HM3KO and MNT-1 were higher than those in primary cultured normal human epithelial melanocytes (NHEM, while GFRa1 transcript expression levels were comparable among NHEM, HM3KO and MNT-1. We next showed c-RET and GFRa1 protein expression in HM3KO cells and GDNF-mediated increased levels of their phosphorylated c-RET tyrosine kinase and signal transduction molecules (ERK and AKT sited potentially downstream of c-RET. Taken together with the finding of augmented proliferation of HM3KO cells after GDNF stimulation, our results suggest that GDNF-mediated c-RET kinase activation is associated with the pathogenesis of malignant melanoma.

  1. The mesenchymal stem cells derived from transgenic mice carrying human coagulation factor VIII can correct phenotype in hemophilia A mice.

    Science.gov (United States)

    Wang, Qing; Gong, Xiuli; Gong, Zhijuan; Ren, Xiaoyie; Ren, Zhaorui; Huang, Shuzhen; Zeng, Yitao

    2013-12-20

    Hemophilia A (HA) is an inherited X-linked recessive bleeding disorder caused by coagulant factor VIII (FVIII) deficiency. Previous studies showed that introduction of mesenchymal stem cells (MSCs) modified by FVIII-expressing retrovirus may result in phenotypic correction of HA animals. This study aimed at the investigation of an alternative gene therapy strategy that may lead to sustained FVIII transgene expression in HA mice. B-domain-deleted human FVIII (hFVIIIBD) vector was microinjected into single-cell embryos of wild-type mice to generate a transgenic mouse line, from which hFVIIIBD-MSCs were isolated, followed by transplantation into HA mice. RT-PCR and real-time PCR analysis demonstrated the expression of hFVIIIBD in multi-organs of recipient HA mice. Immunohistochemistry showed the presence of hFVIIIBD positive staining in multi-organs of recipient HA mice. ELISA indicated that plasma hFVIIIBD level in recipient mice reached its peak (77 ng/mL) at the 3rd week after implantation, and achieved sustained expression during the 5-week observation period. Plasma FVIII activities of recipient HA mice increased from 0% to 32% after hFVIIIBD-MSCs transplantation. APTT (activated partial thromboplastin time) value decreased in hFVIIIBD-MSCs transplanted HA mice compared with untreated HA mice (45.5 s vs. 91.3 s). Our study demonstrated an effective phenotypic correction in HA mice using genetically modified MSCs from hFVIIIBD transgenic mice. Copyright © 2013. Published by Elsevier Ltd.

  2. Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.

    Science.gov (United States)

    Cooper, Caitlin A; Maga, Elizabeth A; Murray, James D

    2015-08-01

    Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

  3. Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP.

    Science.gov (United States)

    Coon, Charles I; Fiering, Steven; Gaudet, Justin; Wyatt, Colby A; Brinckerhoff, Constance E

    2009-09-01

    Matrix metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5'-GGAA-3'(2G allele), and which is an ETS binding site. Compared to the 1G allele (5'-GAA-3'), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity. Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the lac Z (E.coli ss-galactosidase) gene. We measured the relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ss-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.

  4. Presence of subclinical infection in gene-targeted human prion protein transgenic mice exposed to atypical bovine spongiform encephalopathy.

    Science.gov (United States)

    Wilson, Rona; Dobie, Karen; Hunter, Nora; Casalone, Cristina; Baron, Thierry; Barron, Rona M

    2013-12-01

    The transmission of bovine spongiform encephalopathy (BSE) to humans, leading to variant Creutzfeldt-Jakob disease has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health. Until recently, TSE disease in cattle was thought to be caused by a single agent strain, BSE, also known as classical BSE, or BSE-C. However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. To model the risk to human health, we previously inoculated these two forms of atypical BSE (BASE and BSE-H) into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP) (HuTg) but were unable to detect any signs of TSE pathology in these mice. However, despite the absence of TSE pathology, upon subpassage of some BASE-challenged HuTg mice, a TSE was observed in recipient gene-targeted bovine PrP Tg (Bov6) mice but not in HuTg mice. Disease transmission from apparently healthy individuals indicates the presence of subclinical BASE infection in mice expressing human PrP that cannot be identified by current diagnostic methods. However, due to the lack of transmission to HuTg mice on subpassage, the efficiency of mouse-to-mouse transmission of BASE appears to be low when mice express human rather than bovine PrP.

  5. Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus.

    Directory of Open Access Journals (Sweden)

    Guangyu Zhao

    Full Text Available The Middle East Respiratory Syndrome Coronavirus (MERS-CoV causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. For the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of MERS-CoV infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against MERS-CoV infection. In this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hDPP4, the receptor for MERS-CoV. After intranasal inoculation with MERS-CoV, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. In addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. Importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. Taken together, this study characterizes the tropism of MERS-CoV upon infection. Importantly, this hDPP4-expressing transgenic mouse model will be applicable for studying the pathogenesis of MERS-CoV infection and investigating the efficacy of vaccines and antiviral agents designed to combat MERS-CoV infection.

  6. Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664.

    Science.gov (United States)

    Galvan, Veronica; Gorostiza, Olivia F; Banwait, Surita; Ataie, Marina; Logvinova, Anna V; Sitaraman, Sandhya; Carlson, Elaine; Sagi, Sarah A; Chevallier, Nathalie; Jin, Kunlin; Greenberg, David A; Bredesen, Dale E

    2006-05-02

    The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-C31, but whether this C-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.

  7. Low CD4/CD8 T-cell ratio associated with inflammatory arthropathy in human T-cell leukemia virus type I Tax transgenic mice.

    Directory of Open Access Journals (Sweden)

    Takeo Ohsugi

    Full Text Available BACKGROUND: Human T-cell leukemia virus type I (HTLV-1 can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATL as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP. A transgenic mouse that expresses HTLV-1 Tax also develops T-cell leukemia/lymphoma and an inflammatory arthropathy that resembles rheumatoid arthritis. The aim of this study was to identify the primary T-cell subsets involved in the development of arthropathy in Tax transgenic mice. PRINCIPAL FINDINGS: By 24 months of age, Tax transgenic mice developed severe arthropathy with a cumulative incidence of 22.8%. The pathological findings of arthropathy in Tax transgenic mice were similar to those seen in human rheumatoid arthritis or mouse models of rheumatoid arthritis, with synovial proliferation and a positive rheumatoid factor. Before the onset of spontaneous arthropathy, young and old Tax transgenic mice were not sensitive to collagen and did not develop arthritis after immunization with type II collagen. The arthropathic Tax transgenic mice showed a significantly decreased proportion of splenic CD4(+ T cells, whereas the proportion of splenic CD8(+ T cells was increased. Regulatory T cells (CD4(+CD25(+Foxp3(+ were significantly decreased and CD8(+ T cells that expressed the chemokine receptor CCR4 (CD8(+CCR4(+ were significantly increased in arthropathic Tax transgenic mice. The expression of tax mRNA was strong in the spleen and joints of arthropathic mice, with a 40-fold increase compared with healthy transgenic mice. CONCLUSIONS: Our findings reveal that Tax transgenic mice develop rheumatoid-like arthritis with proliferating synovial cells in the joints; however, the proportion of different splenic T-cell subsets in these mice was completely different from other commonly used animal models of rheumatoid arthritis. The crucial T-cell subsets in arthropathic Tax transgenic mice appear to resemble

  8. High concentration of human lactoferrin in milk of rhLf-transgenic cows relieves signs of bovine experimental Staphylococcus chromogenes intramammary infection.

    Science.gov (United States)

    Simojoki, Heli; Hyvönen, Paula; Orro, Toomas; Pyörälä, Satu

    2010-08-15

    Six transgenic cows producing recombinant human lactoferrin (rhLf) in their milk and five normal cows at the same lactation stage were experimentally infected with Staphylococcus chromogenes to study the effect of a high concentration of lactoferrin in milk. Coagulase-negative staphylococci such as S. chromogenes have become very common as agents causing mild or subclinical mastitis. All transgenic cows became infected but showed no clinical signs, unlike the control cows, which developed mild clinical mastitis. Transgenic cows eliminated bacteria faster from the quarters than did the controls. Local clinical signs were milder, and the inflammatory reaction assessed by NAGase activity in the milk and by the concentration of milk amyloid A was lower in the transgenic cows. The mild response probably reflected the rapid elimination of bacteria. The milk concentration of rhLf remained constant throughout the study period, but the total concentration of bovine lactoferrin in the milk peaked in both groups at 46h post-challenge. Three cows, all in the control group, exhibited systemic acute phase response as increased concentrations of serum amyloid A in the blood circulation. Transgenic cows with a high concentration of human lactoferrin in their milk seemed to be protected from clinical disease and from prolonged inflammatory reaction, but not from experimental intramammary infection induced by S. chromogenes.

  9. Serologic analysis of anti-porcine endogenous retroviruses immune responses in humans after ex vivo transgenic pig liver perfusion.

    Science.gov (United States)

    Xu, Hui; Sharma, Ajay; Okabe, Jeannine; Cui, Cunqi; Huang, Liping; Wei, Yuan Yuan; Wan, Hua; Lei, Ying; Logan, John S; Levy, Marlon F; Byrne, Guerard W

    2003-01-01

    Improvements in xenotransplantation may significantly increase the availability of organs for human transplantation. The use of porcine organs, however, has raised concern about possible transmission of porcine endogenous retroviruses (PERV) to the recipients. The authors developed monoclonal antibodies specific to the PERV Gag viral product and show that these antibodies can detect PERV antigen under a variety of assay conditions, including enzyme linked immunosorbent assay (ELISA), Western blot, and immunofluorescence staining methods. Two patients in fulminant hepatic failure were treated by extracorporeal perfusion using transgenic porcine livers before receiving orthotopic liver transplants. Despite the use of immune suppression that allowed survival of the allograft, these patients both showed a strong immune response to the xenograft suggesting a largely intact capability to mount a humoral immune response. However, analysis of patient serum samples over a 3 to 4 year period has showed no evidence of an immune response to PERV antigens, suggesting a lack of PERV infection.

  10. Keratinocyte-specific stat3 heterozygosity impairs development of skin tumors in human papillomavirus 8 transgenic mice.

    Science.gov (United States)

    De Andrea, Marco; Rittà, Massimo; Landini, Manuela M; Borgogna, Cinzia; Mondini, Michele; Kern, Florian; Ehrenreiter, Karin; Baccarini, Manuela; Marcuzzi, Gian Paolo; Smola, Sigrun; Pfister, Herbert; Landolfo, Santo; Gariglio, Marisa

    2010-10-15

    Human papillomaviruses (HPV) of the genus β are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of β-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell-autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3(+/-):HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3(+/+):HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3(+/-):HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer.

  11. TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.

    Science.gov (United States)

    Wu, Yong; Gao, Tieli; Wang, Xiaolin; Hu, Youjin; Hu, Xuyun; Hu, Zhiqing; Pang, Jialun; Li, Zhuo; Xue, Jinfeng; Feng, Mai; Wu, Lingqian; Liang, Desheng

    2014-03-28

    Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.

  12. Sub-chronic (13-week) oral toxicity study in rats with recombinant human lactoferrin produced in the milk of transgenic cows

    NARCIS (Netherlands)

    Appel, M.J.; Veen, H.A. van; Vietsch, H.; Salaheddine, M.; Nuijens, J.H.; Ziere, B.; Loos, F. de

    2006-01-01

    The oral toxicity of recombinant human lactoferrin (rhLF) produced in the milk of transgenic cows was investigated in Wistar rats by daily administration via oral gavage for 13 consecutive weeks, 7 days per week. The study used four groups of 20 rats/sex/dose. The control group received physiologica

  13. A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk

    NARCIS (Netherlands)

    Bischoff, Rainer; Degryse, E.; Perraud, F.; Dalemans, W.; Ali-Hadji, D.; Thepot, D.; Devinoy, E.; Houdebine, L.M.; Pavirani, A.

    1992-01-01

    We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5' flanking sequence could potentially confer, in vivo, high level expression of reporter genes. Transgenic mice were generated expressing a variant of human alpha 1-antitrypsin, which has inhibitory ac

  14. Interferon-gamma (IFN-γ-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

    Directory of Open Access Journals (Sweden)

    Shahid Husain

    Full Text Available We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC death using this model. RGC function was measured by pattern electroretinograms (ERGs in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03 and RGC numbers (37%, p = 0.0001 were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174 ± 27% vs. HLA-A2: 100%; p = 0.04. The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189 ± 11% vs. HLA-A2: 100%; p = 0.023. Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer, which was substantially reduced in IFN-γ ((-/- knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs.

  15. Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

    Science.gov (United States)

    Acosta, Jamie Rae; Goldsbury, Claire; Winnick, Claire; Badrock, Andrew P; Fraser, Stuart T; Laird, Angela S; Hall, Thomas E; Don, Emily K; Fifita, Jennifer A; Blair, Ian P; Nicholson, Garth A; Cole, Nicholas J

    2014-01-01

    FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

  16. Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

    Directory of Open Access Journals (Sweden)

    Jamie Rae Acosta

    Full Text Available FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS, a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

  17. Homing of GAD65 specific autoimmunity and development of insulitis requires expression of both DQ8 and human GAD65 in transgenic mice

    Science.gov (United States)

    Elagin, Raya B.; Balijepalli, Sadguna; Diacovo, Maria J.; Baekkeskov, Steinunn; Jaume, Juan C.

    2009-01-01

    MHC-class II genes determine susceptibility in human type-1 diabetes. In their context, presentation of target antigen(s) results in autoimmunity and β-cell destruction. An animal model, in which human β-cell autoantigen(s) are presented to effector-cells in the context of human MHC-class II diabetes susceptibility genes, would be desirable for studying molecular mechanisms of disease and developing antigen-specific immune-interventions. We report the development of antigen-specific insulitis in double-transgenic mice carrying the HLA-DQ8 diabetes susceptibility haplotype and expressing the human autoantigen GAD65 in pancreatic β-cells. Immunization with human GAD65 cDNA resulted in severe insulitis and low antibody levels in double-transgenic mice while control mice were mostly insulitis free. CFA/protein immunization resulted in high antibody levels and modest insulitis. Pancreatic lymphocytic infiltration progressed through stages (exocrine pancreas followed by peri and intra-insulitis). Adoptive transfer of splenocytes from DNA-immunized mice resulted in development of insulitis in recipient transgenics. Our results show that immunization with a clinically relevant, type-1 diabetes human autoantigen, in a humanized genetic setting, results in the development of an immune response that homes to islets of Langerhans. This animal model will facilitate studies of autoimmunity to GAD65 in the context of HLA-DQ8, and development of methods to induce tolerance and prevent insulitis. PMID:19289270

  18. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Directory of Open Access Journals (Sweden)

    Giulia Breveglieri

    2015-01-01

    Full Text Available Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6 carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a the transgenic integration region is located in mouse chromosome 7; (b the expression of the transgene is tissue specific; (c as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin αmu-globin2/βhu-globin2 and, more importantly, (d the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia.

  19. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Science.gov (United States)

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  20. Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.

    Science.gov (United States)

    Ma, Julian K-C; Drossard, Jürgen; Lewis, David; Altmann, Friedrich; Boyle, Julia; Christou, Paul; Cole, Tom; Dale, Philip; van Dolleweerd, Craig J; Isitt, Valerie; Katinger, Dietmar; Lobedan, Martin; Mertens, Hubert; Paul, Mathew J; Rademacher, Thomas; Sack, Markus; Hundleby, Penelope A C; Stiegler, Gabriela; Stoger, Eva; Twyman, Richard M; Vcelar, Brigitta; Fischer, Rainer

    2015-10-01

    Although plant biotechnology has been widely investigated for the production of clinical-grade monoclonal antibodies, no antibody products derived from transgenic plants have yet been approved by pharmaceutical regulators for clinical testing. In the Pharma-Planta project, the HIV-neutralizing human monoclonal antibody 2G12 was expressed in transgenic tobacco (Nicotiana tabacum). The scientific, technical and regulatory demands of good manufacturing practice (GMP) were addressed by comprehensive molecular characterization of the transgene locus, confirmation of genetic and phenotypic stability over several generations of transgenic plants, and by establishing standard operating procedures for the creation of a master seed bank, plant cultivation, harvest, initial processing, downstream processing and purification. The project developed specifications for the plant-derived antibody (P2G12) as an active pharmaceutical ingredient (API) based on (i) the guidelines for the manufacture of monoclonal antibodies in cell culture systems; (ii) the draft European Medicines Agency Points to Consider document on quality requirements for APIs produced in transgenic plants; and (iii) de novo guidelines developed with European national regulators. From the resulting process, a GMP manufacturing authorization was issued by the competent authority in Germany for transgenic plant-derived monoclonal antibodies for use in a phase I clinical evaluation. Following preclinical evaluation and ethical approval, a clinical trial application was accepted by the UK national pharmaceutical regulator. A first-in-human, double-blind, placebo-controlled, randomized, dose-escalation phase I safety study of a single vaginal administration of P2G12 was carried out in healthy female subjects. The successful completion of the clinical trial marks a significant milestone in the commercial development of plant-derived pharmaceutical proteins. © 2015 Society for Experimental Biology, Association of

  1. Human cryptochrome-1 confers light independent biological activity in transgenic Drosophila correlated with flavin radical stability.

    Directory of Open Access Journals (Sweden)

    Jacqueline Vieira

    Full Text Available Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome--1 (HsCRY1 confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.

  2. Intracellular Aβ pathology and early cognitive impairments in a transgenic rat overexpressing human amyloid precursor protein: a multidimensional study.

    Science.gov (United States)

    Iulita, M Florencia; Allard, Simon; Richter, Luise; Munter, Lisa-Marie; Ducatenzeiler, Adriana; Weise, Christoph; Do Carmo, Sonia; Klein, William L; Multhaup, Gerhard; Cuello, A Claudio

    2014-06-05

    Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-β (Aβ) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular Aβ remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the Aβ pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal Aβ in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble Aβ42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. Aβ38, Aβ39, Aβ40 and Aβ42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular Aβ may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (Aβ, APP and CTFs) of which a considerable component is Aβ; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.

  3. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Directory of Open Access Journals (Sweden)

    Shigeyuki Yamaguchi

    Full Text Available The production of cells capable of expressing gene(s of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  4. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    Science.gov (United States)

    Yamaguchi, Shigeyuki; Kazuki, Yasuhiro; Nakayama, Yuji; Nanba, Eiji; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2011-02-24

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.

  5. Kidneys From α1,3-Galactosyltransferase Knockout/Human Heme Oxygenase-1/Human A20 Transgenic Pigs Are Protected From Rejection During Ex Vivo Perfusion With Human Blood.

    Science.gov (United States)

    Ahrens, Hellen E; Petersen, Björn; Ramackers, Wolf; Petkov, Stoyan; Herrmann, Doris; Hauschild-Quintern, Janet; Lucas-Hahn, Andrea; Hassel, Petra; Ziegler, Maren; Baars, Wiebke; Bergmann, Sabine; Schwinzer, Reinhard; Winkler, Michael; Niemann, Heiner

    2015-07-01

    Multiple modifications of the porcine genome are required to prevent rejection after pig-to-primate xenotransplantation. Here, we produced pigs with a knockout of the α1,3-galactosyltransferase gene (GGTA1-KO) combined with transgenic expression of the human anti-apoptotic/anti-inflammatory molecules heme oxygenase-1 and A20, and investigated their xenoprotective properties. The GGTA1-KO/human heme oxygenase-1 (hHO-1)/human A20 (hA20) transgenic pigs were produced in a stepwise approach using zinc finger nuclease vectors targeting the GGTA1 gene and a Sleeping Beauty vector coding for hA20. Two piglets were analyzed by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and sequencing. The biological function of the genetic modifications was tested in a (51)Chromium release assay and by ex vivo kidney perfusions with human blood. Disruption of the GGTA1 gene by deletion of few basepairs was demonstrated in GGTA1-KO/hHO-1/hA20 transgenic pigs. The hHO-1 and hA20 mRNA expression was confirmed by quantitative reverse-transcription polymerase chain reaction. Ex vivo perfusion of 2 transgenic kidneys was feasible for the maximum experimental time of 240 minutes without symptoms of rejection. Results indicate that GGTA1-KO/hHO-1/hA20 transgenic pigs are a promising model to alleviate rejection and ischemia-reperfusion damage in porcine xenografts and could serve as a background for further genetic modifications toward the production of a donor pig that is clinically relevant for xenotransplantation.

  6. Expression of recombinant human lysozyme in transgenic chicken promotes the growth of Bifidobacterium in the intestine and improves postnatal growth of chicken

    OpenAIRE

    Wang, Hai; Wu, Hongping; Wang, Kejun; Cao, Zhichen; Yu, Kun; Lian, Ling; Lian, Zhengxing

    2016-01-01

    Lysozyme is one kind of antimicrobial proteins and often used as feed additive which can defend against pathogenic bacteria and enhance immune function of animals. In this study, we have injected the lentiviral vector expressing recombinant human lysozyme (rhLZ) gene into the blastoderm of chicken embryo to investigate the effect of recombinant human lysozyme on postnatal intestinal microbiota distribution and growth performance of chicken. Successfully, we generated 194 transgenic chickens i...

  7. Development of transgenic cloned pig models of skin inflammation by DNA transposon-directed ectopic expression of human β1 and α2 integrin.

    Directory of Open Access Journals (Sweden)

    Nicklas Heine Staunstrup

    Full Text Available Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to

  8. Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.

    Science.gov (United States)

    Park, Sol Ji; Cho, Bumrae; Koo, Ok Jae; Kim, Hwajung; Kang, Jung Taek; Hurh, Sunghoon; Kim, Su Jin; Yeom, Hye Jung; Moon, Joonho; Lee, Eun Mi; Choi, Ji Yei; Hong, Ju Ho; Jang, Goo; Hwang, Joing-Ik; Yang, Jaeseok; Lee, Byeong Chun; Ahn, Curie

    2014-06-01

    Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury

  9. Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions.

    Science.gov (United States)

    Ma, Biao; Osborn, Michael J; Avis, Suzanne; Ouisse, Laure-Hélène; Ménoret, Séverine; Anegon, Ignacio; Buelow, Roland; Brüggemann, Marianne

    2013-12-31

    Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3' end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes.

  10. Valproic acid confers functional pluripotency to human amniotic fluid stem cells in a transgene-free approach.

    Science.gov (United States)

    Moschidou, Dafni; Mukherjee, Sayandip; Blundell, Michael P; Drews, Katharina; Jones, Gemma N; Abdulrazzak, Hassan; Nowakowska, Beata; Phoolchund, Anju; Lay, Kenneth; Ramasamy, T Selvee; Cananzi, Mara; Nettersheim, Daniel; Sullivan, Mark; Frost, Jennifer; Moore, Gudrun; Vermeesch, Joris R; Fisk, Nicholas M; Thrasher, Adrian J; Atala, Anthony; Adjaye, James; Schorle, Hubert; De Coppi, Paolo; Guillot, Pascale V

    2012-10-01

    Induced pluripotent stem cells (iPSCs) with potential for therapeutic applications can be derived from somatic cells via ectopic expression of a set of limited and defined transcription factors. However, due to risks of random integration of the reprogramming transgenes into the host genome, the low efficiency of the process, and the potential risk of virally induced tumorigenicity, alternative methods have been developed to generate pluripotent cells using nonintegrating systems, albeit with limited success. Here, we show that c-KIT+ human first-trimester amniotic fluid stem cells (AFSCs) can be fully reprogrammed to pluripotency without ectopic factors, by culture on Matrigel in human embryonic stem cell (hESC) medium supplemented with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). The cells share 82% transcriptome identity with hESCs and are capable of forming embryoid bodies (EBs) in vitro and teratomas in vivo. After long-term expansion, they maintain genetic stability, protein level expression of key pluripotency factors, high cell-division kinetics, telomerase activity, repression of X-inactivation, and capacity to differentiate into lineages of the three germ layers, such as definitive endoderm, hepatocytes, bone, fat, cartilage, neurons, and oligodendrocytes. We conclude that AFSC can be utilized for cell banking of patient-specific pluripotent cells for potential applications in allogeneic cellular replacement therapies, pharmaceutical screening, and disease modeling.

  11. Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia.

    Directory of Open Access Journals (Sweden)

    Michihiro Yoshida

    Full Text Available Lysophosphatidic acid (LPA acts on LPA2 receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA2 attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA2 alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA2 in intestinal epithelial cells (IECs under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG human LPA2; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA2 colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA2 compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA2 overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA2 alone can lead to intestinal dysplasia.

  12. Transgenic expression of human INS gene in Ins1/Ins2 double knockout mice leads to insulin underproduction and diabetes in some male mice.

    Science.gov (United States)

    Karaca, Melis; Durel, Béatrice; Languille, Laëtitia; Lamotte, Luciane; Tourrel-Cuzin, Cécile; Leroux, Loïc; Abou Sleymane, Gretta; Saint-Just, Susan; Bucchini, Danielle; Ktorza, Alain; Joshi, Rajiv L

    2007-01-01

    We have generated transgenic mouse lines expressing exclusively a human INS transgene on an Ins1/Ins2 double knockout (mIKO) background. The transgene expression was driven by either a 4000 bp or a 353 bp promoter. These transgenic lines, designated mIKO:INS4000 and mIKO:INS353, were viable and fertile. Determination of the amounts of insulin transcripts and total pancreatic insulin content revealed relative insulin underproduction in both lines, from birth to adulthood. Total pancreatic insulin stores in mIKO:INS4000 and mIKO:INS353 mice represented only about 50% and 27%, respectively, as compared to wild-type mice. Morphometric analysis of pancreas did not show any compensatory beta-cell hyperplasia. The majority of animals in both lines remained normoglycemic throughout their lives. Nevertheless, glucose tolerance tests revealed glucose intolerance in nearly half of mIKO:INS4000 male mice, likely due to impaired insulin secretion detected in those animals. In addition, a small fraction (2-4%) of male mice in both lines spontaneously developed diabetes with very distinct pathophysiological features. Diabetes was never seen in female animals. The diabetes developed by mIKO:INS353 mice was rapidly lethal, accompanied by a dramatic depletion of pancreatic insulin stores whereas the mIKO:INS4000 diabetic animals could live for several months. This suggests a possible link between the structure of the human INS gene promoter and the type of diabetes developed in these lines.

  13. The in vitro protection of human decay accelerating factor and hDAF/heme oxygenase-1 transgenes in porcine aortic endothelial cells against sera of Formosan macaques.

    Science.gov (United States)

    Tu, C-F; Tai, H-C; Wu, C-P; Ho, L-L; Lin, Y-J; Hwang, C-S; Yang, T-S; Lee, J-M; Tseng, Y-L; Huang, C-C; Weng, C-N; Lee, P-H

    2010-01-01

    To mitigate hyperacute rejection, pigs have been generated with alpha-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF(+), hDAF(+)/hHO-1(+), and hDAF(+)/hHO-1(-) and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to 40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF(+)/hHO-1(+) showed no further protection against effects of MS on transgenic pAEC. Copyright 2010 Elsevier Inc. All rights reserved.

  14. Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

    Science.gov (United States)

    Chakravarthy, Balu; Ito, Shingo; Atkinson, Trevor; Gaudet, Chantal; Ménard, Michel; Brown, Leslie; Whitfield, James

    2014-03-14

    The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aβ1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aβ1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aβ1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice.

  15. Altered heart rate control in transgenic mice carrying the KCNJ6 gene of the human chromosome 21.

    Science.gov (United States)

    Lignon, Jacques M; Bichler, Zoë; Hivert, Bruno; Gannier, François E; Cosnay, Pierre; del Rio, José A; Migliore-Samour, Danièle; Malécot, Claire O

    2008-04-22

    Congenital heart defects (CHD) are common in Down syndrome (DS, trisomy 21). Recently, cardiac sympathetic-parasympathetic imbalance has also been documented in DS adults free of any CHD. The KCNJ6 gene located on human chromosome 21 encodes for the Kir3.2/GIRK2 protein subunits of G protein-regulated K(+) (K(G)) channels and could contribute to this altered cardiac regulation. To elucidate the role of its overexpression, we used homozygous transgenic (Tg(+/+)) mice carrying copies of human KCNJ6. These mice showed human Kir3.2 mRNA expression in the heart and a 2.5-fold increased translation in the atria. Phenotypic alterations were assessed by recording electrocardiogram of urethane anesthetized mice. Chronotropic responses to direct (carbachol) and indirect (methoxamine) muscarinic stimulation were enhanced in Tg(+/+) mice with respect to wild-type (WT) mice. Alternating periods of slow and fast rhythm induced by CCPA (2-chloro-N-cyclopentyl-adenosine) were amplified in Tg(+/+) mice, resulting in a reduced negative chronotropic effect. These drugs reduced the atrial P wave amplitude and area. P wave variations induced by methoxamine and CCPA were respectively increased and reduced in the Tg(+/+) mice, while PR interval and ventricular wave showed no difference between Tg(+/+) and WT. These results indicate that Tg(+/+) mice incorporating the human KCNJ6 exhibit altered Kir3.2 expression and responses to drugs that would activate K(G) channels. Moreover, these altered expression and responses are limited to sino-atrial node and atria that normally express large amounts of K(G) channels. These data suggest that KCNJ6 could play an important role in altered cardiac regulation in DS patients.

  16. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  17. A human FSHB transgene encoding the double N-glycosylation mutant (Asn(7Δ) Asn(24Δ)) FSHβ subunit fails to rescue Fshb null mice.

    Science.gov (United States)

    Wang, Huizhen; Butnev, Vladimir; Bousfield, George R; Kumar, T Rajendra

    2016-05-05

    Follicle-stimulating hormone (FSH) is a gonadotrope-derived heterodimeric glycoprotein. Both the common α- and hormone-specific β subunits contain Asn-linked N-glycan chains. Recently, macroheterogeneous FSH glycoforms consisting of β-subunits that differ in N-glycan number were identified in pituitaries of several species and subsequently the recombinant human FSH glycoforms biochemically characterized. Although chemical modification and in vitro site-directed mutagenesis studies defined the roles of N-glycans on gonadotropin subunits, in vivo functional analyses in a whole-animal setting are lacking. Here, we have generated transgenic mice with gonadotrope-specific expression of either an HFSHB(WT) transgene that encodes human FSHβ WT subunit or an HFSHB(dgc) transgene that encodes a human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, and separately introduced these transgenes onto Fshb null background using a genetic rescue strategy. We demonstrate that the human FSHβ(Asn7Δ 24Δ) double N-glycosylation site mutant subunit, unlike human FSHβ WT subunit, inefficiently combines with the mouse α-subunit in pituitaries of Fshb null mice. FSH dimer containing this mutant FSHβ subunit is inefficiently secreted with very low levels detectable in serum. Fshb null male mice expressing HFSHB(dgc) transgene are fertile and exhibit testis tubule size and sperm number similar to those of Fshb null mice. Fshb null female mice expressing the mutant, but not WT human FSHβ subunit-containing FSH dimer are infertile, demonstrate no evidence of estrus cycles, and many of the FSH-responsive genes remain suppressed in their ovaries. Thus, HFSHB(dgc) unlike HFSHB(WT) transgene does not rescue Fshb null mice. Our genetic approach provides direct in vivo evidence that N-linked glycans on FSHβ subunit are essential for its efficient assembly with the α-subunit to form FSH heterodimer in pituitary. Our studies also reveal that N-glycans on FSHβ subunit are

  18. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Jun Liu

    Full Text Available The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3 in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli, Staphylococcus aureus (S. aureus and Streptococcus agalactiae (S. agalactiae that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05. The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3 and 95.4×10(3 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC in infected glands reached up to 260.4×10(5 and 622.2×10(5 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  19. Anti-bacterial activity of recombinant human β-defensin-3 secreted in the milk of transgenic goats produced by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Jun; Luo, Yan; Ge, Hengtao; Han, Chengquan; Zhang, Hui; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Gao, Mingqing; Zhang, Yong

    2013-01-01

    The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90-111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5-10.5, 21.8-23.0 and 17.3-18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×10(3) and 95.4×10(3) CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×10(5) and 622.2×10(5) cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.

  20. Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells.

    Science.gov (United States)

    Zhu, Haibao; Lau, Cia-Hin; Goh, Sal-Lee; Liang, Qingle; Chen, Can; Du, Shouhui; Phang, Rui-Zhe; Tay, Felix Chang; Tan, Wee-Kiat; Li, Zhendong; Tay, Johan Chin-Kang; Fan, Weimin; Wang, Shu

    2013-10-01

    Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

  1. The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression.

    Science.gov (United States)

    Fang, Lin; Ahn, Jong Kun; Wodziak, Dariusz; Sibley, Eric

    2012-07-01

    Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.

  2. Genetic human prion disease modelled in PrP transgenic Drosophila.

    OpenAIRE

    Thackray, Alana Maureen; Cardova, Alzbeta; Wolf, Hanna; Pradl, Lydia; Vorberg, Ina; Jackson, Walker S.; Bujdoso, Raymond

    2017-01-01

    Inherited human prion diseases, such as FFI and familial CJD (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allo...

  3. Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

    Directory of Open Access Journals (Sweden)

    Yu-Guo Yuan

    2014-01-01

    Full Text Available To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC containing a hybrid goat β-lactoglobulin (BLG promoter/cytomegalovirus (CMV enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT. Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2% from the non-transfected line and five recipients delivered six kids (1.8% from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P<0.05. Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

  4. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variab

  5. 'Transgenous Philosophy': Post-Humanism, Anthropotechnics and the Poetics of Natal Difference

    NARCIS (Netherlands)

    S. Tuinen (Sjoerd)

    2011-01-01

    textabstractIn this paper I investigate Peter Sloterdijk's relation to humanism, especially in its post- Kantian sense of an ideology of Enlightenment based on anthropology. How does an author who writes after Nietzsche's biopolitical challenge of the Übermensch, Heidegger's ontological upgrading of

  6. 'Transgenous Philosophy': Post-Humanism, Anthropotechnics and the Poetics of Natal Difference

    NARCIS (Netherlands)

    S. Tuinen (Sjoerd)

    2011-01-01

    textabstractIn this paper I investigate Peter Sloterdijk's relation to humanism, especially in its post- Kantian sense of an ideology of Enlightenment based on anthropology. How does an author who writes after Nietzsche's biopolitical challenge of the Übermensch, Heidegger's ontological upgrading of

  7. Sabutoclax, a Mcl-1 Antagonist, Inhibits Tumorigenesis in Transgenic Mouse and Human Xenograft Models of Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Roger S. Jackson, II

    2012-07-01

    Full Text Available Resistance to available therapeutic agents has been a common problem thwarting progress in treatment of castrate-resistant and metastatic prostate cancer (PCa. Overexpression of the Bcl-2 family members, including Mcl-1, in PCa cells is known to inhibit intracellular mitochondrial-dependent apoptosis. Here we report the development of a novel transgenic mouse model that spontaneously develops prostatic intraepithelial neoplasia and adenocarcinoma by the inducible, conditional knockout of transforming growth factor β receptor type II in stromal fibroblastic cells (Tgfbr2ColTKO. The Tgfbr2ColTKO prostate epithelia demonstrated down-regulation of luminal and basal differentiation markers, as well as Pten expression and up-regulation of Mcl-1. However, unlike in men, Tgfbr2ColTKO prostates exhibited no regression acutely after castration. The administration of Sabutoclax (BI-97C1, a pan-active Bcl-2 protein family antagonist mediated apoptosis in castrate-resistant PCa cells of Tgfbr2ColTKO mice and human subcutaneous, orthotopic, and intratibial xenograft PCa models. Interestingly, Sabutoclax had little apoptotic effect on benign prostate tissue in Tgfbr2ColTKO and wild-type mice. Sabutoclax was able to block c-Met activation, a critical axis in PCa metastatic progression. Further, Sabutoclax synergistically sensitized PC-3 cells to the cytotoxic effects of docetaxel (Taxotere. Together, these data suggest that Sabutoclax inhibits castrate-resistant PCa alone at the primary and bone metastatic site as well as support sensitivity to docetaxel treatment.

  8. Effects of Neonatal Iron Feeding and Chronic Clioquinol Administration on the Parkinsonian Human A53T Transgenic Mouse.

    Science.gov (United States)

    Billings, Jessica L; Hare, Dominic J; Nurjono, Milawaty; Volitakis, Irene; Cherny, Robert A; Bush, Ashley I; Adlard, Paul A; Finkelstein, David I

    2016-03-16

    Increased nigral iron (Fe) is a cardinal feature of Parkinson's disease, as is the accumulation of aggregates comprising α-synuclein. We used wild-type mice and transgenic mice overexpressing the human A53T mutation to α-synuclein to examine the influence of increased Fe (days 10-17 postpartum) on the parkinsonian development phenotype of these animals (including abnormal nigral Fe levels and deficits in both cell numbers and locomotor activity), and to explore the impact of the Fe chelator clioquinol in the model. Both untreated and Fe-loaded A53T mice showed similar levels of nigral cell loss, though 5 months of clioquinol treatment was only able to prevent the loss in the non-Fe-loaded A53T group. Iron levels in the Fe-loaded A53T mice returned to normal at 8 months, though effects of dopamine denervation remained, demonstrated by limited locomotor activity and sustained neuron loss. These data suggest that Fe exposure during a critical developmental window, combined with the overexpression mutant α-synuclein, presents a disease phenotype resistant to intervention using clioquinol later in life.

  9. Anthrax lethal factor as an immune target in humans and transgenic mice and the impact of HLA polymorphism on CD4+ T cell immunity.

    Science.gov (United States)

    Ascough, Stephanie; Ingram, Rebecca J; Chu, Karen K; Reynolds, Catherine J; Musson, Julie A; Doganay, Mehmet; Metan, Gökhan; Ozkul, Yusuf; Baillie, Les; Sriskandan, Shiranee; Moore, Stephen J; Gallagher, Theresa B; Dyson, Hugh; Williamson, E Diane; Robinson, John H; Maillere, Bernard; Boyton, Rosemary J; Altmann, Daniel M

    2014-05-01

    Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

  10. Anthrax lethal factor as an immune target in humans and transgenic mice and the impact of HLA polymorphism on CD4+ T cell immunity.

    Directory of Open Access Journals (Sweden)

    Stephanie Ascough

    2014-05-01

    Full Text Available Bacillus anthracis produces a binary toxin composed of protective antigen (PA and one of two subunits, lethal factor (LF or edema factor (EF. Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

  11. Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity

    Science.gov (United States)

    Ascough, Stephanie; Ingram, Rebecca J.; Chu, Karen K.; Reynolds, Catherine J.; Musson, Julie A.; Doganay, Mehmet; Metan, Gökhan; Ozkul, Yusuf; Baillie, Les; Sriskandan, Shiranee; Moore, Stephen J.; Gallagher, Theresa B.; Dyson, Hugh; Williamson, E. Diane; Robinson, John H.; Maillere, Bernard; Boyton, Rosemary J.; Altmann, Daniel M.

    2014-01-01

    Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified. PMID:24788397

  12. N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.

    Science.gov (United States)

    Jongen, Susanne P; Gerwig, Gerrit J; Leeflang, Bas R; Koles, Kate; Mannesse, Maurice L M; van Berkel, Patrick H C; Pieper, Frank R; Kroos, Marian A; Reuser, Arnold J J; Zhou, Qun; Jin, Xiaoying; Zhang, Kate; Edmunds, Tim; Kamerling, Johannis P

    2007-06-01

    Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the

  13. Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast.

    Science.gov (United States)

    Zhang, Yujian; Tian, Defeng; Matsuyama, Hironori; Hamazaki, Takashi; Shiratsuchi, Takayuki; Terada, Naohiro; Hook, Derek J; Walters, Michael A; Georg, Gunda I; Hawkinson, Jon E

    2016-04-01

    Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival. © 2016 Society for Laboratory Automation and Screening.

  14. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    Science.gov (United States)

    McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  15. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    Directory of Open Access Journals (Sweden)

    Sandra M McLachlan

    Full Text Available Transgenic mice with the human thyrotropin-receptor (TSHR A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors, regulatory T cell (Treg depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens

  16. A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Abraham Acevedo-Arozena

    2011-09-01

    Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disorder that results in the death of motor neurons in the brain and spinal cord. The disorder generally strikes in mid-life, relentlessly leading to paralysis and death, typically 3–5 years after diagnosis. No effective treatments are available. Up to 10% of ALS is familial, usually autosomal dominant. Several causative genes are known and, of these, mutant superoxide dismutase 1 (SOD1 is by far the most frequently found, accounting for up to 20% of familial ALS. A range of human mutant SOD1 transgenic mouse strains has been produced, and these largely successfully model the human disease. Of these, the most widely used is the SOD1 mouse, which expresses a human SOD1 transgene with a causative G93A mutation. This mouse model is excellent for many purposes but carries up to 25 copies of the transgene and produces a great excess of SOD1 protein, which might affect our interpretation of disease processes. A variant of this strain carries a deletion of the transgene array such that the copy number is dropped to eight to ten mutant SOD1 genes. This ‘deleted’ ‘low-copy’ mouse undergoes a slower course of disease, over many months. Here we have carried out a comprehensive analysis of phenotype, including nerve and muscle physiology and histology, to add to our knowledge of this ‘deleted’ strain and give baseline data for future studies. We find differences in phenotype that arise from genetic background and sex, and we quantify the loss of nerve and muscle function over time. The slowly progressive pathology observed in this mouse strain could provide us with a more appropriate model for studying early-stage pathological processes in ALS and aid the development of therapies for early-stage treatments.

  17. Dissecting genetic requirements of human breast tumorigenesis in a tissue transgenic model of human breast cancer in mice.

    Science.gov (United States)

    Wu, Min; Jung, Lina; Cooper, Adrian B; Fleet, Christina; Chen, Lihao; Breault, Lyne; Clark, Kimberly; Cai, Zuhua; Vincent, Sylvie; Bottega, Steve; Shen, Qiong; Richardson, Andrea; Bosenburg, Marcus; Naber, Stephen P; DePinho, Ronald A; Kuperwasser, Charlotte; Robinson, Murray O

    2009-04-28

    Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.

  18. Primary T-cells from human CD4/CCR5-transgenic rats support all early steps of HIV-1 replication including integration, but display impaired viral gene expression

    Directory of Open Access Journals (Sweden)

    Hermann Volker

    2007-07-01

    Full Text Available Abstract Background In vivo studies on HIV-1 pathogenesis and testing of antiviral strategies have been hampered by the lack of an immunocompetent small animal model that is highly susceptible to HIV-1 infection. Since native rodents are non-permissive, we developed transgenic rats that selectively express the HIV-1 receptor complex, hCD4 and hCCR5, on relevant target cells. These animals display a transient low-level plasma viremia after HIV-1YU-2 infection, demonstrating HIV-1 susceptibility in vivo. However, unlike macrophages, primary CD4 T-cells from double-transgenic animals fail to support viral spread ex vivo. To identify quantitative limitations or absolute blocks in this rodent species, we quantitatively assessed the efficiency of key steps in the early phase of the viral replication cycle in a side-by-side comparison in infected cell lines and primary T-cells from hCD4/hCCR5-transgenic rats and human donors. Results Levels of virus entry, HIV-1 cDNA synthesis, nuclear import, and integration into the host genome were shown to be remarkably similar in cell lines and, where technically accessible, in primary T-cells from both species. In contrast, a profound impairment at the level of early HIV gene expression was disclosed at the single-cell level in primary rat T-cells and most other rat-derived cells. Macrophages were a notable exception, possibly reflecting the unique transcriptional milieu in this evolutionarily conserved target cell of all lentiviruses. Importantly, transient trans-complementation by ex vivo nucleofection with the Tat-interacting protein Cyclin T1 of human origin markedly elevated HIV gene expression in primary rat T-cells. Conclusion This is the first study that has quantitatively determined the efficiency of consecutive steps in the HIV-1 replication cycle in infected primary HIV target cells from a candidate transgenic small animal and compared it to human cells. Unlike cells derived from mice or rabbits, rat

  19. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Pittius, C.W.; Hennighausen, L.; Lee, E.; Westphal, H.; Nicols, E.; Vitale, J.; Gordon, K. (National Institutes of Health, Bethesda, MD (USA))

    1988-08-01

    Whey acidic protein (WAP) is a major whey protein in mouse milk. Its gene is expressed in the lactating mammary gland and is inducible by steroid and peptide hormones. A series of transgenic mice containing a hybrid gene in which human tissue plasminogen activator (tPA) cDNA is under the control of the murine WAP gene promoter had previously been generated. In this study, 21 tissues from lactating and virgin transgenic female mice containing the WAP-tPA hybrid gene were screened for the distribution of murine WAP and human tPA transcripts. Like the endogenous WAP RNA, WAP-tPA RNA was expressed predominantly in mammary gland tissue and appeared to be inducible by lactation. Whereas WAP transcripts were not detected in 22 tissues of virgin mice, low levels of WAP-tPA RNA, which were not modulated during lactation, were found in tongue, kidney, and sublingual gland. These studies demonstrate that the WAP gene promoter can target the expression of a transgene to the mammary gland and that this expression is inducible during lactation.

  20. 转人α-乳清白蛋白奶粉的遗传毒性研究%Genetic toxicity of transgenic human α-lactalbumin powdered milk

    Institute of Scientific and Technical Information of China (English)

    支媛; 刘海波; 耿桂英; 崔文明; 刘珊; 徐海滨

    2013-01-01

    目的:评价转人α-乳清白蛋白奶粉的遗传毒性.方法:以转人α-乳清白蛋白奶粉为受试物,设每皿62、185、556、1 667、5 000μg 5个剂量进行Ames试验,按2.5、5.0和10.0 g/kg进行小鼠骨髓嗜多染红细胞 (polychromatic erythrocytes,PCE)微核试验和精子畸形试验,并设立环磷酰胺阳性对照组和蒸馏水阴性对照组.结果:转人α-乳清白蛋白奶粉各剂量组回变菌落数均未超过溶剂对照菌落数的2倍,亦无剂量-反应关系;各剂量组PCE百分比未少于阴性对照组的20%,微核发生率与阴性对照组比较差异无统计学意义 (P>0.05);各剂量组小鼠精子畸形发生率与阴性对照组比较差异无统计学意义 (P>0.05).结论:在本实验条件下,转人α-乳清白蛋白奶粉未见遗传毒性.%OBJECTIVE: To evaluate genetic toxicity of transgenic human α-lactalbumin powdered milk. METHODS:In Ames test,TA97,TA98,TA100,and TA102 strains were treated by 62,185,556,1 667 and 5 000 μg transgenic human α-lactalbumin powdered milk per plate. In mice bone marrow cell micronucleus test and mice sperm abnormality test,2.5,5.0 and 10.0 g/kg transgenic human α-lactalbumin powdered milk groups,CP positive and water negative control groups were set up. RESULTS:Back mutation colonies of transgenic human α-lactalbumin powdered milk groups did not exceed twice that of the control in Ames test and there was no dose-response relationship. In mice bone marrow cell micronucleus test,no significant difference was found in PCE/RBC and micronucleus rate between transgenic human α-lactalbumin powdered milk-treated groups and negative control group. In mice sperm abnormality test,sperm abnormality rate of transgenic human α-lactalbumin powdered milk-treated groups was lower than that of negative control. CONCLUSION:No genetic toxicity was observed in transgenic human α-lactalbumin powdered milk.

  1. Modulation of human neural stem cell differentiation in Alzheimer (APP23) transgenic mice by phenserine.

    Science.gov (United States)

    Marutle, Amelia; Ohmitsu, Masao; Nilbratt, Mats; Greig, Nigel H; Nordberg, Agneta; Sugaya, Kiminobu

    2007-07-24

    In a previous study, we found that human neural stem cells (HNSCs) exposed to high concentrations of secreted amyloid-precursor protein (sAPP) in vitro differentiated into mainly astrocytes, suggesting that pathological alterations in APP processing during neurodegenerative conditions such as Alzheimer's disease (AD) may prevent neuronal differentiation of HNSCs. Thus, successful neuroplacement therapy for AD may require regulating APP expression to favorable levels to enhance neuronal differentiation of HNSCs. Phenserine, a recently developed cholinesterase inhibitor (ChEI), has been reported to reduce APP levels in vitro and in vivo. In this study, we found reductions of APP and glial fibrillary acidic protein (GFAP) levels in the hippocampus of APP23 mice after 14 days treatment with (+)-phenserine (25 mg/kg) lacking ChEI activity. No significant change in APP gene expression was detected, suggesting that (+)-phenserine decreases APP levels and reactive astrocytes by posttranscription regulation. HNSCs transplanted into (+)-phenserine-treated APP23 mice followed by an additional 7 days of treatment with (+)-phenserine migrated and differentiated into neurons in the hippocampus and cortex after 6 weeks. Moreover, (+)-phenserine significantly increased neuronal differentiation of implanted HNSCs in hippocampal and cortical regions of APP23 mice and in the CA1 region of control mice. These results indicate that (+)-phenserine reduces APP protein in vivo and increases neuronal differentiation of HNSCs. Combination use of HNSC transplantation and treatment with drugs such as (+)-phenserine that modulate APP levels in the brain may be a useful tool for understanding mechanisms regulating stem cell migration and differentiation during neurodegenerative conditions in AD.

  2. Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Georgeta Crivat

    Full Text Available The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4, the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM. We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to

  3. Transgenic expression of soluble human CD5 enhances experimentally-induced autoimmune and anti-tumoral immune responses.

    Directory of Open Access Journals (Sweden)

    Rafael Fenutría

    Full Text Available CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EμTg, expressing a circulating soluble form of human CD5 (shCD5 as a decoy to impair membrane-bound CD5 function. These shCD5EμTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE, as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma. This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+, and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EμTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens.

  4. Inhibition of human UGT2B7 gene expression in transgenic mice by the constitutive androstane receptor.

    Science.gov (United States)

    Yueh, M F; Mellon, P L; Tukey, R H

    2011-06-01

    The xenobiotic receptors, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) regulate and alter the metabolism of xenobiotic substrates. Among the 19 functional UDP-glucuronosyltransferases (UGTs) in humans, UGT2B7 is involved in the metabolism of many structurally diverse xenobiotics and plays an important role in the clearance and detoxification of many therapeutic drugs. To examine whether this gene is regulated by CAR and PXR in vivo, transgenic mice expressing the entire UGT2B7 gene (TgUGT2B7) were created. Gene expression profiles revealed that UGT2B7 is differentially expressed in liver, kidney, adipocytes, brain, and estrogen-sensitive tissues, such as ovary and uterus. Liver UGT2B7 expression levels were decreased when TgUGT2B7 mice were treated with the CAR ligand 1,4-b-s-[2-(3,5,-dichloropyridyloxy)] (TCPOBOP) but not the PXR ligand pregnenolone 16α-carbonitrile. Although TCPOBOP decreased the levels of UGT2B7 mRNA in TgUGT2B7 mice, it had no affect on Tg(UGT2B7)Car(-/-) mice, adding support for a CAR-dependent mechanism contributing toward UGT2B7 gene suppression. Expression of promoter constructs in HepG2 cells showed the CAR-dependent inhibition was linked to hepatocyte nuclear factor-4α (HNF4α)-mediated transactivation of the UGT2B7 promoter. The inhibitory effect of CAR on UGT2B7 gene expression was validated in chromatin immunoprecipitation assays in which TCPOBOP treatment blocked HNF4α binding to the UGT2B7 promoter. These results suggest that HNF4α plays an important role in the constitutive expression of hepatic UGT2B7, and CAR acts as a negative regulator by interfering with HNF4α binding activity.

  5. Insulin Stimulates Translocation of Human GLUT4 to the Membrane in Fat Bodies of Transgenic Drosophila melanogaster

    Science.gov (United States)

    Crivat, Georgeta; Lizunov, Vladimir A.; Li, Caroline R.; Stenkula, Karin G.; Zimmerberg, Joshua; Cushman, Samuel W.; Pick, Leslie

    2013-01-01

    The fruit fly Drosophila melanogaster is an excellent model system for studies of genes controlling development and disease. However, its applicability to physiological systems is less clear because of metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of relevance to diabetes and other diseases but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in ‘diabetic’ flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues as seen in mammals is unclear and depends upon whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. Here we asked whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. Transgenic Drosophila expressing human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues, were generated. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence microscopy (TIRFM). We found that fat body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin. They suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant, high levels of sugar. Finally, these studies are the first to use TIRFM to monitor insulin

  6. A native outer membrane vesicle vaccine confers protection against meningococcal colonization in human CEACAM1 transgenic mice.

    Science.gov (United States)

    Pajon, Rolando; Buckwalter, Carolyn M; Johswich, Kay O; Gray-Owen, Scott D; Granoff, Dan M

    2015-03-10

    The effect of protein-based meningococcal vaccines on prevention of nasopharyngeal colonization has been difficult to investigate experimentally because a reliable animal colonization model did not exist. Human CEACAM1 transgenic mice, which can be colonized by meningococci, were immunized IP with one of two meningococcal native outer membrane vesicle (NOMV) vaccines prepared from mutants with attenuated endotoxin (lpxL1 knockout) and over-expressed sub-family B Factor H-binding proteins (FHbp). Animals were challenged intranasally two weeks after the third dose with wild-type strain H44/76, or were treated IP with anti-NOMV serum before and during the bacterial challenge. The NOMV-1 vaccine, prepared from the serogroup B H44/76 mutant, elicited ∼40-fold higher serum bactericidal antibody titers against the wild-type H44/76 challenge strain than the NOMV-2 vaccine prepared from a heterologous serogroup W mutant strain with different PorA and FHbp amino acid sequence variants. Compared to aluminum hydroxide-immunized control mice, the efficacy for prevention of any H44/76 colonization was 93% (95% confidence interval, 52-99, P<0.0001) for the NOMV-1 vaccine, and 19% (-3-36, P=0.23) for NOMV-2. NOMV-2-vaccinated mice had a 5.6-fold decrease in geometric mean CFU of bacteria per animal in tracheal washes compared to control mice (P=0.007). The efficacy of passive administration of serum from NOMV-1-vaccinated mice to immunologically naïve mice against colonization was 44% (17-61; P=0.002). Both NOMV vaccines protected against meningococcal colonization but there was greater protection by the NOMV-1 vaccine with antigens matched with the challenge strain. Meningococcal vaccines that target protein antigens have potential to decrease colonization. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Mehdi Sharifi Tabar

    2015-10-01

    Full Text Available Objective: Genetic modification of human embryonic stem cells (hESCs is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI into the target cell line, however, there are few reports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for transient expression of green fluorescent protein in two genetically different hESC lines, Royan H5 (XX and Royan H6 (XY, as well as human foreskin fibroblasts (hFF. For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively, were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after transfection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs was conducted to confirm stable integration of the transgene. Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 μg and the density of the subjected cells (5×105 and 1×106 cells were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion

  8. Gene expression profile of cervical and skin tissues from human papillomavirus type 16 E6 transgenic mice

    Directory of Open Access Journals (Sweden)

    Lambert PF

    2008-11-01

    Full Text Available Abstract Background Although K14E6 transgenic mice develop spontaneous tumors of the skin epithelium, no spontaneous reproductive tract malignancies arise, unless the transgenic mice were treated chronically with 17β-estradiol. These findings suggest that E6 performs critical functions in normal adult cervix and skin, highlighting the need to define E6-controlled transcriptional programs in these tissues. Methods We evaluated the expression profile of 14,000 genes in skin or cervix from young K14E6 transgenic mice compared with nontransgenic. To identify differentially expressed genes a linear model was implemented using R and the LIMMA package. Two criteria were used to select the set of relevant genes. First a set of genes with a Log-odds ≥ 3 were selected. Then, a hierarchical search of genes was based on Log Fold Changes. Results Microarray analysis identified a total of 676 and 1154 genes that were significantly up and down-regulated, respectively, in skin from K14E6 transgenic mice. On the other hand, in the cervix from K14E6 transgenic mice we found that only 97 and 252 genes were significantly up and down-regulated, respectively. One of the most affected processes in the skin from K14E6 transgenic mice was the cell cycle. We also found that skin from transgenic mice showed down-regulation of pro-apoptotic genes and genes related to the immune response. In the cervix of K14E6 transgenic mice, we could not find affected any gene related to the cell cycle and apoptosis pathways but did observe alterations in the expression of immune response genes. Pathways such as angiogenesis, cell junction and epidermis development, also were altered in their gene expression profiles in both tissues. Conclusion Expression of the HPV16 E6 oncoprotein in our model alters expression of genes that fell into several functional groups providing insights into pathways by which E6 deregulate cell cycle progression, apoptosis, the host resistance to infection

  9. Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuanyuan [Department of Pharmaceutics, Qingdao University of Science and Technology, 53 Zhengzhou Road, P.O. Box 70, Qingdao 266042 (China); Liu, Junhong, E-mail: liujh@qust.edu.cn [Department of Pharmaceutics, Qingdao University of Science and Technology, 53 Zhengzhou Road, P.O. Box 70, Qingdao 266042 (China)

    2011-05-15

    Transgenic alfalfa plants simultaneously expressing human CYP2E1 and glutathione S-transferase (GST) were generated from hypocotyl segments by the use of an Agrobacterium transformation system for the phytoremediation of the mixed contaminated soil with heavy metals and organic pollutants. The transgenic alfalfa plants were screened by a combination of kanamycin resistance, PCR, GST and CYP2E1 activity and Western blot analysis. The capabilities of mixed contaminants (heavy metals-organic compounds) resistance of pKHCG transgenic alfalfa plants became markedly increased compared with the transgenic alfalfa plants expressing single gene (GST or CYP2E1) and the non-transgenic control plants. The pKHCG alfalfa plants exhibited strong resistance towards the mixtures of cadmium (Cd) and trichloroethylene (TCE) that were metabolized by the introduced GST and CYP2E1 in combination. Our results show that the pKHCG transgenic alfalfa plants have good potential for phytoremediation because they have cross-tolerance towards the complex contaminants of heavy metals and organic pollutants. Therefore, these transgenic alfalfa plants co-expressing GST and human P450 CDNAs may have a great potential for phytoremediation of mixed environmental contaminants.

  10. P301S mutant human tau transgenic mice manifest early symptoms of human tauopathies with dementia and altered sensorimotor gating.

    Directory of Open Access Journals (Sweden)

    Hiroki Takeuchi

    Full Text Available Tauopathies are neurodegenerative disorders characterized by the accumulation of abnormal tau protein leading to cognitive and/or motor dysfunction. To understand the relationship between tau pathology and behavioral impairments, we comprehensively assessed behavioral abnormalities in a mouse tauopathy model expressing the human P301S mutant tau protein in the early stage of disease to detect its initial neurological manifestations. Behavioral abnormalities, shown by open field test, elevated plus-maze test, hot plate test, Y-maze test, Barnes maze test, Morris water maze test, and/or contextual fear conditioning test, recapitulated the neurological deficits of human tauopathies with dementia. Furthermore, we discovered that prepulse inhibition (PPI, a marker of sensorimotor gating, was enhanced in these animals concomitantly with initial neuropathological changes in associated brain regions. This finding provides evidence that our tauopathy mouse model displays neurofunctional abnormalities in prodromal stages of disease, since enhancement of PPI is characteristic of amnestic mild cognitive impairment, a transitional stage between normal aging and dementia such as Alzheimer's disease (AD, in contrast with attenuated PPI in AD patients. Therefore, assessment of sensorimotor gating could be used to detect the earliest manifestations of tauopathies exemplified by prodromal AD, in which abnormal tau protein may play critical roles in the onset of neuronal dysfunctions.

  11. Human Islet Amyloid Polypeptide Transgenic Mice: In Vivo and Ex Vivo Models for the Role of hIAPP in Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    J. W. M. Höppener

    2008-01-01

    Full Text Available Human islet amyloid polypeptide (hIAPP, a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2. To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process.

  12. Expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene under control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice.

    Science.gov (United States)

    Burkov, I A; Serova, I A; Battulin, N R; Smirnov, A V; Babkin, I V; Andreeva, L E; Dvoryanchikov, G A; Serov, O L

    2013-10-01

    Expression of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene under the control of the 5'-regulatory sequence of the goat alpha-S1-casein gene with and without a matrix attachment region (MAR) element from the Drosophila histone 1 gene was studied in four and eight transgenic mouse lines, respectively. Of the four transgenic lines carrying the transgene without MAR, three had correct tissues-specific expression of the hGM-CSF gene in the mammary gland only and no signs of cell mosaicism. The concentration of hGM-CSF in the milk of transgenic females varied from 1.9 to 14 μg/ml. One line presented hGM-CSF in the blood serum, indicating ectopic expression. The values of secretion of hGM-CSF in milk of 6 transgenic lines carrying the transgene with MAR varied from 0.05 to 0.7 μg/ml, and two of these did not express hGM-CSF. Three of the four examined animals from lines of this group showed ectopic expression of the hGM-CSF gene, as determined by RT-PCR and immunofluorescence analyses, as well as the presence of hGM-CSF in the blood serum. Mosaic expression of the hGM-CSF gene in mammary epithelial cells was specific to all examined transgenic mice carrying the transgene with MAR but was never observed in the transgenic mice without MAR. The mosaic expression was not dependent on transgene copy number. Thus, the expected "protective or enhancer effect" from the MAR element on the hGM-CSF gene expression was not observed.

  13. Regulation of blood pressure, oxidative stress and AT1R by high salt diet in mutant human dopamine D5 receptor transgenic mice.

    Science.gov (United States)

    Liu, Xing; Wang, Wenjie; Chen, Wei; Jiang, Xiaoliang; Zhang, Yanrong; Wang, Zihao; Yang, Jian; Jones, John E; Jose, Pedro A; Yang, Zhiwei

    2015-06-01

    Humans have dopamine D5 receptors (hD5R) with single-nucleotide polymorphisms and a diminished function. We generated hD5(F173L) cDNA that has a decreased response to D5R agonist-mediated increase in cAMP production and increased production of reactive oxygen species, relative to wild-type hD5R (hD5(WT)) cDNA expressed in Chinese hamster ovary cells. To investigate the role of hD5(F173L) in the pathogenesis of salt-sensitive hypertension, we generated transgenic mice overexpressing hD5(F173L) or hD5(WT) and fed them normal (0.8% NaCl) or high (4% NaCl) salt diet. On normal salt diet, the blood pressure, and renal NADPH oxidase activity and angiotensin type 1 receptor (AT1R) expression were higher in hD5(F173L) than hD5(WT) transgenic mice. After 2 weeks on high salt diet, the blood pressure and renal NADPH oxidase activity, but not AT1R expression, were increased in hD5(F173L) but not in hD5(WT) transgenic mice. Candesartan, an AT1R antagonist, decreased the blood pressure and NADPH oxidase activity in hD5(F173L) but not in hD5(WT) transgenic mice. We suggest that the ability of the hD5R to negatively regulate the renal NADPH oxidase activity and AT1R function may have important implications in the pathogenesis of salt-sensitive blood pressure. However, the mechanisms involved in regulating the balance of renal D5R and AT1R function in the oxidative stress-mediated salt-sensitive blood pressure remain to be determined.

  14. Human Leukocyte Antigen-DQ8 Transgenic Mice: A Model to Examine the Toxicity of Aerosolized Staphylococcal Enterotoxin B

    Science.gov (United States)

    2004-11-30

    inser- tion of HLA-DQ10301 and HLA-DQ10302 gene fragments created the HLA-DQ8 transgenic mice. The C57BL/6 embryos were inserted into (C57BL/6 DBA...enterotoxin B. Toxicol. Pathol. 31:373–378. 31. Stevens, D. L. 2000. Streptococcal toxic shock syndrome associated with necrotizing fasciitis . Annu. Rev. Med

  15. Prion-like propagation of human brain-derived alpha-synuclein in transgenic mice expressing human wild-type alpha-synuclein.

    Science.gov (United States)

    Bernis, Maria E; Babila, Julius T; Breid, Sara; Wüsten, Katharina Annick; Wüllner, Ullrich; Tamgüney, Gültekin

    2015-11-26

    Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases that are characterized by the intracellular accumulation of alpha-synuclein containing aggregates. Recent increasing evidence suggests that Parkinson's disease and MSA pathology spread throughout the nervous system in a spatiotemporal fashion, possibly by prion-like propagation of alpha-synuclein positive aggregates between synaptically connected areas. Concurrently, intracerebral injection of pathological alpha-synuclein into transgenic mice overexpressing human wild-type alpha-synuclein, or human alpha-synuclein with the familial A53T mutation, or into wild-type mice causes spreading of alpha-synuclein pathology in the CNS. Considering that wild-type mice naturally also express a threonine at codon 53 of alpha-synuclein, it has remained unclear whether human wild-type alpha-synuclein alone, in the absence of endogenously expressed mouse alpha-synuclein, would support a similar propagation of alpha-synuclein pathology in vivo. Here we show that brain extracts from two patients with MSA and two patients with probable incidental Lewy body disease (iLBD) but not phosphate-buffered saline induce prion-like spreading of pathological alpha-synuclein after intrastriatal injection into mice expressing human wild-type alpha-synuclein. Mice were sacrificed at 3, 6, and 9 months post injection and analyzed neuropathologically and biochemically. Mice injected with brain extracts from patients with MSA or probable iLBD both accumulated intraneuronal inclusion bodies, which stained positive for phosphorylated alpha-synuclein and appeared predominantly within the injected brain hemisphere after 6 months. After 9 months these intraneuronal inclusion bodies had spread to the contralateral hemisphere and more rostral and caudal areas. Biochemical analysis showed that brains of mice injected with brain extracts from patients with MSA and probable iLBD contained hyperphosphorylated alpha

  16. Conditional transgenic mice expressing C-terminally truncated human α-synuclein (αSyn119 exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons

    Directory of Open Access Journals (Sweden)

    Flint Beal M

    2009-07-01

    Full Text Available Abstract Background Missense mutations and multiplications of the α-synuclein gene cause autosomal dominant familial Parkinson's disease (PD. α-Synuclein protein is also a major component of Lewy bodies, the hallmark pathological inclusions of PD. Therefore, α-synuclein plays an important role in the pathogenesis of familial and sporadic PD. To model α-synuclein-linked disease in vivo, transgenic mouse models have been developed that express wild-type or mutant human α-synuclein from a variety of neuronal-selective heterologous promoter elements. These models exhibit a variety of behavioral and neuropathological features resembling some aspects of PD. However, an important deficiency of these models is the observed lack of robust or progressive nigrostriatal dopaminergic neuronal degeneration that is characteristic of PD. Results We have developed conditional α-synuclein transgenic mice that can express A53T, E46K or C-terminally truncated (1–119 human α-synuclein pathological variants from the endogenous murine ROSA26 promoter in a Cre recombinase-dependent manner. Using these mice, we have evaluated the expression of these α-synuclein variants on the integrity and viability of nigral dopaminergic neurons with age. Expression of A53T α-synuclein or truncated αSyn119 selectively in nigrostriatal pathway dopaminergic neurons for up to 12 months fails to precipitate dopaminergic neuronal loss in these mice. However, αSyn119 expression in nigral dopaminergic neurons for up to 12 months causes a marked reduction in the levels of striatal dopamine and its metabolites together with other subtle neurochemical alterations. Conclusion We have developed and evaluated novel conditional α-synuclein transgenic mice with transgene expression directed selectively to nigrostriatal dopaminergic neurons as a potential new mouse model of PD. Our data support the pathophysiological relevance of C-terminally truncated α-synuclein species in vivo. The

  17. Transgenic farm animals: applications in agriculture and biomedicine.

    Science.gov (United States)

    Yang, X; Tian, X C; Dai, Y; Wang, B

    2000-01-01

    During the last decade, tremendous progress has been made in the area of transgenic farm animals. While there are many important transgenic farm animal applications in agriculture, funding has been very limited and progress has been rather slow in this area. Encouragingly, the potential applications of transgenic farm animals as bioreactors for producing human therapeutic proteins and as organ donors for transplantations in humans have attracted vast funding from the private sectors. Several transgenic animal products are already in various phases of clinical trials. Estimates are, that in the near future, the worlds demands on human pharmaceutical proteins may largely be met by transgenic farm animals. While there are still major challenges ahead in the area of xenotransplantation using transgenic animal organs, transgenic tissues or cells have demonstrated promising results as a potential tool for gene therapy. Recent development on cloning, embryonic stem cells and alternative transgenic methods may further expand the transgenic applications in both agriculture and biomedicine.

  18. Spontaneous destructive periodontitis and skeletal bone damage in transgenic mice carrying a human shared epitope-coding HLA-DRB1 allele

    Science.gov (United States)

    Gehlot, Prashasnika; Volk, Sarah L; Rios, Hector F; Jepsen, Karl J; Holoshitz, Joseph

    2016-01-01

    Objective Shared epitope (SE)-coding DRB1 alleles are associated with bone erosion in several diseases, including rheumatoid arthritis (RA) and periodontal disease (PD), but the underlying mechanism is unknown. We have recently identified the SE as an osteoclast-activating ligand. To better understand the biological effects of the SE in vivo, here we sought to determine whether it can facilitate spontaneous bone damage in naïve mice. Methods 3-month old naïve transgenic mice that carry the human SE-coding allele DRB1*04:01, or a SE-negative allele DRB1*04:02 were studied. Bone tissues were analysed by micro-CT, and the tooth-supporting tissues were studied by histology, immunohistochemistry and immunofluorescence. Serum biomarkers were determined by ELISA. Results Transgenic mice expressing the SE-coding DRB1*04:01 allele, but not mice carrying the SE-negative allele DRB1*04:02, showed spontaneous PD associated with interleukin (IL)-17 overabundance and periostin disruption. Mandibular bone volumetric and mineralisation parameters were significantly lower in SE-positive mice, and alveolar bone resorption was significantly increased in these mice. SE-positive mice also had more slender tibiae, and their marrow, cortical and total areas were lower than those of SE-negative mice. Additionally, significantly increased serum IL-17, tumour necrosis factor-α and osteoprotegrin levels were found in SE-positive mice, while their receptor activator of nuclear factor κ-B ligand levels were significantly lower. Conclusions A human SE-coding allele increases the propensity to spontaneous bone-destructive periodontal inflammation and skeletal bone damage in transgenic mice. These findings provide new insights into the previously documented but poorly understood association of the SE with accelerated bone erosion in RA and several other human diseases. PMID:27933212

  19. A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G

    Science.gov (United States)

    Jain, Sudhir; Vinukonda, Govindaiah; Fiering, Steven N.; Kumar, Ashok

    2008-01-01

    The human angiotensinogen (hAGT) gene contains an A/G polymorphism at −217, and frequency of −217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant −217A almost always occurs with −532T, −793A, and −1074T, whereas variant −217G almost always occurs with −532C, −793G, and −1074G. Since allele −6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main haplotypes, −6A:−217A (AA) and −6A:−217G (AG). To understand the role of these haplotypes on hAGT gene expression and on blood pressure regulation in an in vivo situation, we have generated double transgenic mice containing human renin gene and either AA or AG haplotype of the hAGT gene using knock-in strategy at the hypoxanthine phosphoribosyltransferase locus. We show here that 1) hAGT mRNA level is increased in the liver by 60% and in the kidney by 40%; and 2) plasma AGT level is increased by ∼40%, and plasma angiotensin II level is increased by ∼50% in male double transgenic mice containing AA haplotype of the hAGT gene compared with the AG haplotype. In addition, systolic blood pressure is increased by 8 mmHg in transgenic mice containing the AA haplotype compared with the AG haplotype. This is the first report to show the effect of polymorphisms in the promoter of a human gene on its transcription in an in vivo situation that ultimately leads to an increase in blood pressure. PMID:18945948

  20. COMPARISON TRANSGENIC AND NON-TRANSGENIC MILK QUALITY

    Directory of Open Access Journals (Sweden)

    Peter Chrenek

    2012-02-01

    Full Text Available Transgenic founder rabbits carrying a gene construct consisting of a 2.5 kb murine whey acidic protein promoter (mWAP, 7.2 kb of the human clotting factor VIII (hFVIII cDNA and 4.6 kb of 3’ flanking sequences of mWAP gene were crossed for five generations. Transgenic females showed high level of recombinant hFVIII (rhFVIII mRNA expression in biopsed mammary gland tissues. The presence of the mWAP-hFVIII transgene in rabbit genome and secretion of rhFVIII into milk of transgenic females (F1, F2, F3, F4 and F5 generation did not have any adverse phenotypic effect on milk quality.

  1. A review of piscine islet xenotransplantation using wild-type tilapia donors and the production of transgenic tilapia expressing a "humanized" tilapia insulin.

    Science.gov (United States)

    Wright, James R; Yang, Hua; Hyrtsenko, Olga; Xu, Bao-You; Yu, Weiming; Pohajdak, Bill

    2014-01-01

    Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long-term normoglycemia and provide human-like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T-cell-dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co-stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.

  2. Transgenic animal bioreactors.

    Science.gov (United States)

    Houdebine, L M

    2000-01-01

    involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases.

  3. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Margarita Chumarina

    2017-03-01

    Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

  4. Effect on peripheral nerve regeneration by transgene in vivo with human insulin-like growth factor-1

    Institute of Scientific and Technical Information of China (English)

    Jiaxiang Gu; Yufa Wang; Shanhe Dai

    2006-01-01

    BACKGROUND: Human insulin-like growth factor (hIGF-1) has been successful in treating peripheral nerve injury, but it is still unclear whether hlGF-1 after transgene in vivo has the effect on promoting the regeneration of peripheral nerve. OBJECTIVE: To observe the effect of hlGF-1 on the regeneration of peripheral nerve by transgene in vivo with electrophysiology, histological morphology and ultromicro morphology. DESIGN: A univariate design. SETTINGS: Jilin Institute of Surgery, China-Japan Friendship Hospital Affiliated to Jilin University; School of Basic Medical Sciences, Jilin University. MATERIALS: Thirty male adult Wistar rats of grade Ⅱ, weighing 200-250 g, were provided by the Animal Experimental Center of Jilin University [certification number: SCXK-(Ji)20030001]. The rats were raised in the environment at the temperature of 25 ℃ and humidity of 70%. All the rats were randomly divided into hlGF-1-treated group, treatment control group and blank control group, 10 rats in each group. Positive liposomes (mass concentration of 2 g/L) and pcDNA3.1 (mass concentration of 1 g/L) were purchased from Beijing Yuanpinghao Company; pcDNAhlGF-1 (mass concentration of 1 g/L) was provided by Dr. Shen from the School of Public Health of Jilin University. The liposomes were mixed with plasmids with the mass ratio of 1.5 to 10.Operative microscope was made by Jiangsu Zhenjiang Microsurgical Instrument Factory; EMB-5304K electromyogram (EMG) evoked potential meter by Nihon Kohden Corporation. HPIAS-1 000 high-acuity color pathological imaging analytical system (Japan) and JEM-1200EX transmission electron microscope (Japan) were also used. METHODS: The experiments were carried out in Jilin Institute of Surgery from April to June in 2004. ① All the rats were anesthetized, and the right sciatic nerve was exposed, and it was clipped with a clip at 5 mm below the piriform muscle for 3 times, 10 s for each time. The pressed width was 3 mm, and formed as membrane under

  5. In Vivo Determination of Vitamin D Function Using Transgenic Mice Carrying a Human Osteocalcin Luciferase Reporter Gene

    Directory of Open Access Journals (Sweden)

    Tomoko Nakanishi

    2013-01-01

    Full Text Available Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1α,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

  6. Evaluation of 89Zr-rituximab tracer by Cerenkov luminescence imaging and correlation with PET in a humanized transgenic mouse model to image NHL.

    Science.gov (United States)

    Natarajan, Arutselvan; Habte, Frezghi; Liu, Hongguang; Sathirachinda, Ataya; Hu, Xiang; Cheng, Zhen; Nagamine, Claude M; Gambhir, Sanjiv Sam

    2013-08-01

    This research aimed to study the use of Cerenkov luminescence imaging (CLI) for non-Hodgkin's lymphoma (NHL) using 89Zr-rituximab positron emission tomography (PET) tracer with a humanized transgenic mouse model that expresses human CD20 and the correlation of CLI with PET. Zr-rituximab (2.6 MBq) was tail vein-injected into transgenic mice that express the human CD20 on their B cells (huCD20TM). One group (n=3) received 2 mg/kg pre-dose (blocking) of cold rituximab 2 h prior to tracer; a second group (n=3) had no pre-dose (non-blocking). CLI was performed using a cooled charge-coupled device optical imager. We also performed PET imaging and ex vivo studies in order to confirm the in vivo CLI results. At each time point (4, 24, 48, 72, and 96 h), two groups of mice were imaged in vivo and ex vivo with CLI and PET, and at 96 h, organs were measured by gamma counter. huCD20 transgenic mice injected with 89Zr-rituximab demonstrated a high-contrast CLI image compared to mice blocked with a cold dose. At various time points of 4-96 h post-radiotracer injection, the in vivo CLI signal intensity showed specific uptake in the spleen where B cells reside and, hence, the huCD20 biomarker is present at very high levels. The time-activity curve of dose decay-corrected CLI intensity and percent injected dose per gram of tissue of PET uptake in the spleen were increased over the time period (4-96 h). At 96 h, the 89Zr-rituximab uptake ratio (non-blocking vs blocking) counted (mean±standard deviation) for the spleen was 1.5±0.6 for CLI and 1.9±0.3 for PET. Furthermore, spleen uptake measurements (non-blocking and blocking of all time points) of CLI vs PET showed good correlation (R2=0.85 and slope=0.576), which also confirmed the corresponding correlations parameter value (R2=0.834 and slope=0.47) obtained for ex vivo measurements. CLI and PET of huCD20 transgenic mice injected with 89Zr-rituximab demonstrated that the tracer was able to target huCD20-expressing B cells. The in

  7. Transgenic Mice with −6A Haplotype of the Human Angiotensinogen Gene Have Increased Blood Pressure Compared with −6G Haplotype*

    Science.gov (United States)

    Jain, Sudhir; Tillinger, Andrej; Mopidevi, Brahmaraju; Pandey, Varunkumar G.; Chauhan, Chetankumar K.; Fiering, Steven N.; Warming, Soren; Kumar, Ashok

    2010-01-01

    Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at −6, and the −6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with −6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with −6G. We have found that the hAGT gene has three additional SNPs (A/G at −1670, C/G at −1562, and T/G at −1561). Variants −1670A, −1562C, and −1561T almost always occur with −6A, and variants −1670G, −1562G, and −1561G almost always occur with −6G. Therefore, the hAGT gene may be subdivided into either −6A or −6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with −6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with −6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the −6A or −6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing −6A haplotype. Our studies also show that transgenic mice containing −6A haplotype have increased plasma AGT level and increased blood pressure as compared with −6G haplotype. Our studies explain the molecular mechanism involved in association of the −6A allele of the hAGT gene with hypertension. PMID:20978123

  8. Vector-free and transgene-free human iPS cells differentiate into functional neurons and enhance functional recovery after ischemic stroke in mice.

    Directory of Open Access Journals (Sweden)

    Osama Mohamad

    Full Text Available Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.

  9. Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island

    Directory of Open Access Journals (Sweden)

    Antoniou Michael

    2007-09-01

    Full Text Available Abstract Background A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox inducible rtTA2S-M2 Tet-transactivator in transgenic mice. Results An 8 kb genomic fragment from the methylation-free CpG island of the human hnRNPA2B1-CBX3 housekeeping gene locus was tested. In a number of transgenic mouse lines obtained, rtTA2S-M2 expression was detected in many tissues examined. Characterisation of the highest expressing rtTA2S-M2 transgenic mouse line demonstrated Dox-inducible GFP transgene expression in many tissues. Using this line we also show highly sensitive quantitative induction with low doses of Dox of an assayable plasma protein transgene under the control of a Tet Responsive Element (TRE. The utility of this rtTA2S-M2 line for inducible expression in mouse embryos was also demonstrated using a GATA-6 Tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene. Conclusion The ubiquitously expressing rtTA2S-M2 transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mice.

  10. Renin-angiotensin system transgenic mouse model recapitulates pathophysiology similar to human preeclampsia with renal injury that may be mediated through VEGF.

    Science.gov (United States)

    Denney, J Morgan; Bird, Cynthia; Gendron-Fitzpatrick, Annette; Sampene, Emmanuel; Bird, Ian M; Shah, Dinesh M

    2017-03-01

    Using a transgenic cross, we evaluated features of preeclampsia, renal injury and the sFlt1/VEGF changes. Transgenic hAGT and hREN, or wild-type (WT) C57Bl/6 mice were cross-bred: female hAGT × male hREN for preeclampsia (PRE) model and female WT × male WT for pregnant controls (WTP). Samples were collected for plasma VEGF, sFlt1, and urine albumin. Blood pressures (BP) were monitored by telemetry. Vascular reactivity was investigated by wire myography. Kidneys and placenta were immunostained for sFlt1 and VEGF. Eleven PRE and 9 WTP mice were compared. PRE more frequently demonstrated albuminuria, glomerular endotheliosis (80% vs. 11%; P = 0.02), and placental necrosis (60% vs. 0%; P preeclampsia recapitulates human preeclamptic state with high fidelity, and that, vascular adaptation to pregnancy is suggested by declining BPs and reduced vascular response to PE and increased response to acetylcholine. Placental damage with resultant increased release of sFlt1, proteinuria, deficient spiral artery remodeling, and glomerular endotheliosis were observed in this model of PRE. Increased VEGF binding to glomerular endothelial cells in this model of PRE is similar to human PRE and leads us to hypothesize that renal injury in preeclampsia may be mediated through local VEGF. Copyright © 2017 the American Physiological Society.

  11. Centrosomal Nlp is an oncogenic protein that is gene-amplified in human tumors and causes spontaneous tumorigenesis in transgenic mice.

    Science.gov (United States)

    Shao, Shujuan; Liu, Rong; Wang, Yang; Song, Yongmei; Zuo, Lihui; Xue, Liyan; Lu, Ning; Hou, Ning; Wang, Mingrong; Yang, Xiao; Zhan, Qimin

    2010-02-01

    Disruption of mitotic events contributes greatly to genomic instability and results in mutator phenotypes. Indeed, abnormalities of mitotic components are closely associated with malignant transformation and tumorigenesis. Here we show that ninein-like protein (Nlp), a recently identified BRCA1-associated centrosomal protein involved in microtubule nucleation and spindle formation, is an oncogenic protein. Nlp was found to be overexpressed in approximately 80% of human breast and lung carcinomas analyzed. In human lung cancers, this deregulated expression was associated with NLP gene amplification. Further analysis revealed that Nlp exhibited strong oncogenic properties; for example, it conferred to NIH3T3 rodent fibroblasts the capacity for anchorage-independent growth in vitro and tumor formation in nude mice. Consistent with these data, transgenic mice overexpressing Nlp displayed spontaneous tumorigenesis in the breast, ovary, and testicle within 60 weeks. In addition, Nlp overexpression induced more rapid onset of radiation-induced lymphoma. Furthermore, mouse embryonic fibroblasts (MEFs) derived from Nlp transgenic mice showed centrosome amplification, suggesting that Nlp overexpression mimics BRCA1 loss. These findings demonstrate that Nlp abnormalities may contribute to genomic instability and tumorigenesis and suggest that Nlp might serve as a potential biomarker for clinical diagnosis and therapeutic target.

  12. Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

    Directory of Open Access Journals (Sweden)

    Pappu Kameshwari M

    2011-06-01

    Full Text Available Abstract Background Collagens require the hydroxylation of proline (Pro residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H as a posttranslational processing step. Results A recombinant human collagen type I α-1 (rCIα1 with high percentage of hydroxylated prolines (Hyp was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H. Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47% and the native human CIa1 (14.59%, respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1. Conclusions This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.

  13. The Human Leukocyte Antigen (HLA)-B27 Peptidome in Vivo, in Spondyloarthritis-susceptible HLA-B27 Transgenic Rats and the Effect of Erap1 Deletion.

    Science.gov (United States)

    Barnea, Eilon; Melamed Kadosh, Dganit; Haimovich, Yael; Satumtira, Nimman; Dorris, Martha L; Nguyen, Mylinh T; Hammer, Robert E; Tran, Tri M; Colbert, Robert A; Taurog, Joel D; Admon, Arie

    2017-04-01

    HLA-B27 is a class I major histocompatibility (MHC-I) allele that confers susceptibility to the rheumatic disease ankylosing spondylitis (AS) by an unknown mechanism. ERAP1 is an aminopeptidase that trims peptides in the endoplasmic reticulum for binding to MHC-I molecules. ERAP1 shows genetic epistasis with HLA-B27 in conferring susceptibility to AS. Male HLA-B27 transgenic rats develop arthritis and serve as an animal model of AS, whereas female B27 transgenic rats remain healthy. We used large scale quantitative mass spectrometry to identify over 15,000 unique HLA-B27 peptide ligands, isolated after immunoaffinity purification of the B27 molecules from the spleens of HLA-B27 transgenic rats. Heterozygous deletion of Erap1, which reduced the Erap1 level to less than half, had no qualitative or quantitative effects on the B27 peptidome. Homozygous deletion of Erap1 affected approximately one-third of the B27 peptidome but left most of the B27 peptidome unchanged, suggesting the possibility that some of the HLA-B27 immunopeptidome is not processed in the presence of Erap1. Deletion of Erap1 was permissive for the AS-like phenotype, increased mean peptide length and increased the frequency of C-terminal hydrophobic residues and of N-terminal Ala, Ser, or Lys. The presence of Erap1 increased the frequency of C-terminal Lys and Arg, of Glu and Asp at intermediate residues, and of N-terminal Gly. Several peptides of potential interest in AS pathogenesis, previously identified in human cell lines, were isolated. However, rats susceptible to arthritis had B27 peptidomes similar to those of non-susceptible rats, and no peptides were found to be uniquely associated with arthritis. Whether specific B27-bound peptides are required for AS pathogenesis remains to be determined. Data are available via ProteomeXchange with identifier PXD005502.

  14. Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

    Directory of Open Access Journals (Sweden)

    Katrin Spiesberger

    Full Text Available 30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4.With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.

  15. Evidence for the accumulation of Abeta immunoreactive material in the human brain and in transgenic animal models.

    Science.gov (United States)

    Cuello, A Claudio; Allard, Simon; Ferretti, Maria Teresa

    2012-12-10

    In this review we highlight the evidence for an intracellular origin of Abeta (Aβ) amyloid peptides as well as the observations for a pathological accumulation of these peptides in Alzheimer's disease and Down syndrome, as well as in transgenic animal models. We deliberate on the controversy as to whether the intracellular Aβ immunoreactive material is simply an accumulation of unprocessed full length amyloid precursor protein (APP) or a mix of processed APP fragments including Aβ. Finally, we discuss the possible pathological significance of these intracellular APP fragments and the expected future research directions regarding this thought-provoking problem. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. A Method for Producing Transgenic Cells Using a Multi-Integrase System on a Human Artificial Chromosome Vector

    OpenAIRE

    Shigeyuki Yamaguchi; Yasuhiro Kazuki; Yuji Nakayama; Eiji Nanba; Mitsuo Oshimura; Tetsuya Ohbayashi

    2011-01-01

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-spec...

  17. High expression of human se-rum albumin in milk of trans-genic mice directed by the goat b-casein gene promoter region

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    We have constructed a mammary gland expression vector that contained the goat b-casein gene promoter, 5′upstream regulatory region, exons 1, 2, intron 1 as well as the human serum albumin (hALB) mini-gene (including the full-long sequences of hALB cDNA and its intron 1). Injec-tion of the vector into mouse tail veins showed that the re-combinant construct was expressed only in mammary glands. The vector was microinjected into the mouse fertilized eggs, followed by transferring the eggs into the foster mice. 33 F0 mice were obtained. Of the 33, 8 mice (5♀, 3♂) were trans-genic with hALB gene integration identified by PCR as well as Southern blot hybridization. The integration rate was 24.2% (8/33). Western blot analysis showed that 3 female transgenic mice had hALB expression in their milk. The hALB contents in milk reached 3.54, 0.21 and 3.03 g/L, re-spectively.

  18. Expression of a partially deleted gene of human type II procollagen (COL2A1) in transgenic mice produces a chondrodysplasia

    Energy Technology Data Exchange (ETDEWEB)

    Vandenberg, P.; Khillan, J.S.; Prockop, D.J.; Helminen, H.; Kontusaari, S.; Ala-Kokko, L. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1991-09-01

    A minigene version of the human gene for type II procollagen (COL2AI) was prepared that lacked a large central region containing 12 of the 52 exons and therefore 291 of the 1523 codons of the gene. The construct was modeled after sporadic in-frame deletions of collagen genes that cause synthesis of shortened pro{alpha} chains that associate with normal pro{alpha} chains and thereby cause degradation of the shortened and normal pro{alpha} chains through a process called procollagen suicide. The gene construct was used to prepare five lines of transgenic mice expressing the minigene. A large proportion of the mice expressing the minigene developed a phenotype of a chondrodysplasia with dwarfism, short and thick limbs, a short snout, a cranial bulge, a cleft palate, and delayed mineralization of bone. A number of mice died shortly after birth. Microscopic examination of cartilage revealed decreased density and organization of collagen fibrils. In cultured chondrocytes from the transgenic mice, the minigene was expressed as shortened pro{alpha}1(II) chains that were disulfide-linked to normal mouse pro{alpha}1(II) chains. Therefore, the phenotype is probably explained by depletion of the endogenous mouse type II procollagen through the phenomenon of procollagen suicide.

  19. Fumaric acid esters can block pro-inflammatory actions of human CRP and ameliorate metabolic disturbances in transgenic spontaneously hypertensive rats.

    Directory of Open Access Journals (Sweden)

    Jan Šilhavý

    Full Text Available Inflammation and oxidative stress have been implicated in the pathogenesis of metabolic disturbances. Esters of fumaric acid, mainly dimethyl fumarate, exhibit immunomodulatory, anti-inflammatory, and anti-oxidative effects. In the current study, we tested the hypothesis that fumaric acid ester (FAE treatment of an animal model of inflammation and metabolic syndrome, the spontaneously hypertensive rat transgenically expressing human C-reactive protein (SHR-CRP, will ameliorate inflammation, oxidative stress, and metabolic disturbances. We studied the effects of FAE treatment by administering Fumaderm, 10 mg/kg body weight for 4 weeks, to male SHR-CRP. Untreated male SHR-CRP rats were used as controls. All rats were fed a high sucrose diet. Compared to untreated controls, rats treated with FAE showed significantly lower levels of endogenous CRP but not transgenic human CRP, and amelioration of inflammation (reduced levels of serum IL6 and TNFα and oxidative stress (reduced levels of lipoperoxidation products in liver, heart, kidney, and plasma. FAE treatment was also associated with lower visceral fat weight and less ectopic fat accumulation in liver and muscle, greater levels of lipolysis, and greater incorporation of glucose into adipose tissue lipids. Analysis of gene expression profiles in the liver with Affymetrix arrays revealed that FAE treatment was associated with differential expression of genes in pathways that involve the regulation of inflammation and oxidative stress. These findings suggest potentially important anti-inflammatory, anti-oxidative, and metabolic effects of FAE in a model of inflammation and metabolic disturbances induced by human CRP.

  20. Genetically engineered rat gliomas: PDGF-driven tumor initiation and progression in tv-a transgenic rats recreate key features of human brain cancer.

    Science.gov (United States)

    Connolly, Nina P; Stokum, Jesse A; Schneider, Craig S; Ozawa, Tatsuya; Xu, Su; Galisteo, Rebeca; Castellani, Rudolph J; Kim, Anthony J; Simard, J Marc; Winkles, Jeffrey A; Holland, Eric C; Woodworth, Graeme F

    2017-01-01

    Previously rodent preclinical research in gliomas frequently involved implantation of cell lines such as C6 and 9L into the rat brain. More recently, mouse models have taken over, the genetic manipulability of the mouse allowing the creation of genetically accurate models outweighed the disadvantage of its smaller brain size that limited time allowed for tumor progression. Here we illustrate a method that allows glioma formation in the rat using the replication competent avian-like sarcoma (RCAS) virus / tumor virus receptor-A (tv-a) transgenic system of post-natal cell type-specific gene transfer. The RCAS/tv-a model has emerged as a particularly versatile and accurate modeling technology by enabling spatial, temporal, and cell type-specific control of individual gene transformations and providing de novo formed glial tumors with distinct molecular subtypes mirroring human GBM. Nestin promoter-driven tv-a (Ntv-a) transgenic Sprague-Dawley rat founder lines were created and RCAS PDGFA and p53 shRNA constructs were used to initiate intracranial brain tumor formation. Tumor formation and progression were confirmed and visualized by magnetic resonance imaging (MRI) and spectroscopy. The tumors were analyzed using histopathological and immunofluorescent techniques. All experimental animals developed large, heterogeneous brain tumors that closely resembled human GBM. Median survival was 92 days from tumor initiation and 62 days from the first point of tumor visualization on MRI. Each tumor-bearing animal showed time dependent evidence of malignant progression to high-grade glioma by MRI and neurological examination. Post-mortem tumor analysis demonstrated the presence of several key characteristics of human GBM, including high levels of tumor cell proliferation, pseudopalisading necrosis, microvascular proliferation, invasion of tumor cells into surrounding tissues, peri-tumoral reactive astrogliosis, lymphocyte infiltration, presence of numerous tumor

  1. Transcription-dependent silencing of inducible convergent transgenes in transgenic mice

    Directory of Open Access Journals (Sweden)

    Calero-Nieto Fernando J

    2010-01-01

    Full Text Available Abstract Background Silencing of transgenes in mice is a common phenomenon typically associated with short multi-copy transgenes. We have investigated the regulation of the highly inducible human granulocyte-macrophage colony-stimulating-factor gene (Csf2 in transgenic mice. Results In the absence of any previous history of transcriptional activation, this transgene was expressed in T lineage cells at the correct inducible level in all lines of mice tested. In contrast, the transgene was silenced in a specific subset of lines in T cells that had encountered a previous episode of activation. Transgene silencing appeared to be both transcription-dependent and mediated by epigenetic mechanisms. Silencing was accompanied by loss of DNase I hypersensitive sites and inability to recruit RNA polymerase II upon stimulation. This pattern of silencing was reflected by increased methylation and decreased acetylation of histone H3 K9 in the transgene. We found that silenced lines were specifically associated with a single pair of tail-to-tail inverted repeated copies of the transgene embedded within a multi-copy array. Conclusions Our study suggests that epigenetic transgene silencing can result from convergent transcription of inverted repeats which can lead to silencing of an entire multi-copy transgene array. This mechanism may account for a significant proportion of the reported cases of transgene inactivation in mice.

  2. A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

    Directory of Open Access Journals (Sweden)

    Lu Ying

    2004-04-01

    Full Text Available Abstract Background TGM1(transglutaminase 1 is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. Methods In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. Results In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the

  3. Expression of the human apolipoprotein A-I gene in transgenic mice alters high density lipoprotein (HDL) particle size distribution and diminishes selective uptake of HDL cholesteryl esters

    Energy Technology Data Exchange (ETDEWEB)

    Chajekshaul, T.; Hayek, T.; Walsh, A.; Breslow, J.L. (Rockefeller University, New York, NY (USA))

    1991-08-01

    Transgenic mice carrying the human apolipoprotein (apo) A-I gene (HuAITg mice) were used to examine the effects of overexpression of the human gene on high density lipoprotein (HDL) particle size distribution and metabolism. On a chow diet, control mice had HDL cholesterol and apo A-I levels of 49 {plus minus} 2 and 137 {plus minus} 12 mg/dl of plasma, respectively. HuAITg mice had HDL cholesterol, human apo A-I, and mouse apo A-I levels of 88 {plus minus} 2, 255 {plus minus} 19, and 16 {plus minus} 2 mg/dl, respectively. Nondenaturing gradient gel electrophoresis revealed control mouse plasma HDL to be primarily monodisperse with a particle diameter of 10.2 nm, whereas HuAITg mouse plasma HDL was polydisperse with particles of diameter 11.4, 10.2, and 8.7 nm, which correspond in size to human HDL1, HDL2, and HDL3, respectively. In vivo turnover studies of HDL labeled with (3H)cholesteryl linoleyl ether and 125I-apo A-I were performed. In control animals, the fractional catabolic rate (FCR) for HDL cholesteryl ester was significantly more than the apo A-I FCR. In the HuAITg mice, the HDL cholesteryl ester FCR was the same as the apo A-I FCR. There were no significant differences between control and HuAITg animals in the sites of tissue removal of HDL cholesteryl ester, with the liver extracting most of the injected radioactivity. Control and HuAITg animals had comparable liver and intestinal cholesterol synthesis and LDL FCR. In conclusion, HuAITg mice have principally human and not mouse apo A-I in their plasma. This apparently causes a change in HDL particle size distribution in the transgenic mice to one resembling the human pattern. The replacement of mouse by human apo A-I also apparently causes the loss of the selective uptake pathway of HDL cholesteryl esters present in control mice.

  4. The past, present and future of transgenic bioreactors.

    Science.gov (United States)

    Drohan, W N

    1997-07-01

    Hybrid genes can control the tissue-specific synthesis of human proteins in transgenic animals. Thus, it is now possible to produce proteins of biomedical value in the body fluids or cells of transgenic livestock. In fact, the first transgenically produced protein, antithrombin III, is now in clinical trials and others will soon follow.

  5. A transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins through N-glycosylation.

    Science.gov (United States)

    Hu, Jia-Biao; Zhang, Peng; Wang, Mei-Xian; Zhou, Fang; Niu, Yan-Shan; Miao, Yun-Gen

    2012-08-01

    Glycoproteins have been implicated in a wide variety of important biochemical and biological functions, including protein stability, immune function, enzymatic function, cellular adhesion and others. Unfortunately, there is no therapeutic protein produced in insect system to date, due to the expressed glycoproteins are paucimannosidic N-glycans, rather than the complex, terminally sialylated N-glycans in mammalian cells. In this paper, we cloned the necessary genes in glycosylation of mammalian cells, such as N-acetylglucosaminyltransferase II (Gn-TII), galactosyltransferases (Gal-Ts), 2,6-Sial-T (ST6 GalII)and 2,3-Sial-T (ST3GalIII), and transformed them to silkworm genome of BmN cell line through transgenesis to establish a transgenic Bm cell line of piggyBac transposon-derived targeting expression of humanized glycoproteins. The study supplied a new insect cell line which is practically to produce "bisected" complex N-glycans like in mammalian cells.

  6. Phenotype of transgenic mice carrying a very low copy number of the mutant human G93A superoxide dismutase-1 gene associated with amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Jeffrey S Deitch

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1. There is variability in sporadic ALS as well as FALS where even within the same family some siblings with the same mutation do not manifest disease. A transgenic (Tg mouse model of FALS containing 25 copies of the mutant human SOD1 gene demonstrates motor neuron pathology and progressive weakness similar to ALS patients, leading to death at approximately 130 days. The onset of symptoms and survival of these transgenic mice are directly related to the number of copies of the mutant gene. We report the phenotype of a very low expressing (VLE G93A SOD1 Tg carrying only 4 copies of the mutant G93ASOD1 gene. While weakness can start at 9 months, only 74% of mice 18 months or older demonstrate disease. The VLE mice show decreased motor neurons compared to wild-type mice as well as increased cytoplasmic translocation of TDP-43. In contrast to the standard G93A SOD1 Tg mouse which always develops motor weakness leading to death, not all VLE animals manifested clinical disease or shortened life span. In fact, approximately 20% of mice older than 24 months had no motor symptoms and only 18% of VLE mice older than 22 months reached end stage. Given the variable penetrance of clinical phenotype, prolonged survival, and protracted loss of motor neurons the VLE mouse provides a new tool that closely mimics human ALS. This tool will allow the study of pathologic events over time as well as the study of genetic and environmental modifiers that may not be causative, but can exacerbate or accelerate motor neuron disease.

  7. Transgenic agriculture and environmental indicators

    Directory of Open Access Journals (Sweden)

    Denize Dias de Carvalho

    2006-12-01

    Full Text Available Despite the rapid diffusion of transgenic crops, there are still few environmental impact studies capable of supplying a conclusive scientific response in regard to its technical and economic advantages and disadvantages. Prospective scenarios were elaborated to assist environmental impact assessment, using techniques derived from SWOT (Strength, Weakness, Opportunity, Threat analysis and the DPSIR (Driving Force – human activity, Pressure, State, Impact, Response model, to evaluate the environmental indicators and the relationship between them. Control and management actions were identified, searching the integration of aspects related to the biotechnology applied to transgenic processes, biodiversity, biosafety and intellectual property. It was demonstrated that the DPSIR model is, in fact, an instrument for integrated environmental assessment and the application of the proposed methodology resulted in favorable indicators to the adoption of transgenic agriculture. The elaborated scenarios are useful to develop an Environmental Management System (EMS to agriculture.

  8. Evaluation of the efficiency of human immune system reconstitution in NSG mice and NSG mice containing a human HLA.A2 transgene using hematopoietic stem cells purified from different sources.

    Science.gov (United States)

    Patton, John; Vuyyuru, Raja; Siglin, Amanda; Root, Michael; Manser, Tim

    2015-07-01

    Severely immunodeficient mice such as the NOD/SCID/IL2rγ(null) (NSG) strain can be engrafted with human hematopoietic stem cells (HSCs), resulting in chimeric mice containing many components of the human immune system (Human Immune System mice or HIS mice). HIS mice can both support the replication of and recapitulate much of the immunological response to a variety of pathogens, including ones with strict human tropism, such as HIV-1. In an effort to develop a better mouse model for human infectious pathogen infection and possible immune resolution, we compared the human immune system reconstitution of NSG mice following injection with human CD34(+) HSCs purified from either fetal liver (FL) or umbilical cord blood (UCB). We analyzed reconstitution in standard NSG mice as well as a derivative of these mice containing an HLA.A2 encoding transgene (NSG.A2). HSCs from both sources effectively reconstituted hematopoietic lineages when injected into NSG mice. In marked contrast, total CD45(+) human hematopoietic cells in NSG.A2 mice were well reconstituted by HSCs from UCB but very poorly by HSCs purified from FL. Moreover, the reconstitution of T cell lineages in NSG.A2 mice by HSCs from UCB was inferior to that obtained using NSG mice. We also found that FL CD34(+) HSCs contain a much higher percentage of cells with a phenotype consistent with primitive progenitors than UCB HSCs. We discuss possible explanations for the influence of the HLA.A2 transgene on hematopoietic reconstitution using the two sources of HSCs.

  9. Neuroanatomy and transgenic technologies

    Science.gov (United States)

    This is a short review that introduces recent advances of neuroanatomy and transgenic technologies. The anatomical complexity of the nervous system remains a subject of tremendous fascination among neuroscientists. In order to tackle this extraordinary complexity, powerful transgenic technologies a...

  10. Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype.

    Directory of Open Access Journals (Sweden)

    Isobel J Whitehouse

    Full Text Available The cellular prion protein (PrPC has been proposed to play an important role in the pathogenesis of Alzheimer's disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5. Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production.

  11. [Features of Immunophenotypes and Characteristics of Molecular Biology and Cellular Genetics of AML Patients with CD4 and CD7 Expression].

    Science.gov (United States)

    Liu, Tie-Qiang; Huang, Shan; Yao, Bo; Liu, Zhi-Qing; Yu, Chang-Lin; Qiao, Jian-Hui; Sun, Qi-Yun; Hu, Kai-Xun; Huang, Ya-Jing; Zhang, Rui; Li, Yu-Fang; Bai, Juan; Sun, Yu-Jing; Li, Bing-Xia; Wang, Dong-Mei; Wang, Yi; Guo, Mei

    2016-12-01

    To explore the features of immunophenotypes and the characteristics of molecular biology and cellular genetics of AML patients with CD7 and CD4 expression. The immunophenotypical markers of AML cells were detected by multiple parameter flow cytometry; the expression of WT1, MDK, ETO, PML-RaRa and BCR-ABL were detected by RT-PCR; and cellular features were analyzed by R-band in 304 patients. The patients were divided into three groups according to their immunophenotypes: AML with CD7 expression (CD7 group), AML with CD4 expression(CD4 group) and AML without CD7 and CD4 expression (common AML group). The expression rate and level of HLA-DR in CD7 group were higher than those in the common AML group, and the expression rate of CD33 and CD34 was higher than that in the other two groups. The expression rate and level of CD15, CD64 in the CD4 group were higher than those in the other 2 groups, and the expression rate and level of CD33 were higher than those in the common AML group. WT1 expression in the CD7 group was lower than that in the common AML group. PML-RaRa was not detected in the CD7 group. AML with co-expression of CD4 or CD7 showed more normal karyotype. (15;17) was not found in AML with CD7 expression. AML cells with CD7 expression originate from precursor cells and are blocked in the early phase of hematological development; AML cells with CD4 expression originate from more mature stage of hematological devevelopment and with CD33, CD64 and CD15 high expression; AML cells with CD7 and CD4 expression are characterized by no-specific change of cellular genetics. According to the expression level and intesity of CD4 and CD7, and together with other specific lineage markers, the MRD in AML patients can be quantitatively detected.

  12. Expression of the human TSPY gene in the brains of transgenic mice suggests a potential role of this Y chromosome gene in neural functions

    Institute of Scientific and Technical Information of China (English)

    Tatsuo Kido; Stephanie Schubert; J(o)rg Schmidtke; Yun-Fai Chris Lau

    2011-01-01

    The testis specific protein Y-encoded (TSPY) is a member of TSPY/SET/NAPl superfamily, encoded within the gonadoblastoma locus on the Y chromosome. TSPY shares a highly conserved SET/NAP-domain responsible for protein-protein interaction among TSPY/SET/NAPl proteins.Accumulating data, so far, support the role of TSPY as the gonadoblastoma gene, involved in germ cell tumorigenesis. The X-chromosome homolog of TSPY, TSPX is expressed in various tissues at both fetal and adult stages, including the brain, and is capable of interacting with the multi-domain adapter protein CASK, thereby influencing the synaptic and transcriptional functions and developmental regulation of CASK in the brain and other neural tissues. Similar to TSPX, we demonstrated that TSPY could interact with CASK at its SET/NAP-domain in cultured cells. Transgenic mice harboring a human TSPY gene and flanking sequences showed specific expression of the human TSPYtransgene in both testis and brain. The neural expression pattern of the human TSPY gene overlapped with those of the endogenous mouse Cask and Tspx gene. Similarly with TSPX, TSPY was co-localized with CASK in neuronal axon fibers in the brain, suggesting a potential role(s) of TSPY in development and/or physiology of the nervous system.

  13. Transcription activator-like effector nuclease (TALEN-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC and neural stem cell (NSC lines.

    Directory of Open Access Journals (Sweden)

    Trevor Cerbini

    Full Text Available Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs and neural stem cells (NSCs. The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

  14. Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.

    Science.gov (United States)

    Cerbini, Trevor; Funahashi, Ray; Luo, Yongquan; Liu, Chengyu; Park, Kyeyoon; Rao, Mahendra; Malik, Nasir; Zou, Jizhong

    2015-01-01

    Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.

  15. 人源脑红蛋白转基因秀丽线虫的制备、鉴定及生理功能分析%Construction, characterization and physiological functions of transgenic C.elegans expressing human neuroglobin

    Institute of Scientific and Technical Information of China (English)

    石锦平; 任长虹; 李媛; 张继业; 范礼斌; 张成岗

    2011-01-01

    目的 以秀丽隐杆线虫为模式生物制备人源脑红蛋白(human neuroglobin,hNgb)转基因线虫,初步探讨外源性hNgb对线虫寿命和产卵率的影响.方法 通过显微注射方法制备hNgb转基因线虫,使用蛋白印迹和免疫组织化学进行鉴定.使用野生型N2和增强型绿色荧光蛋白(EGFP)转基因线虫作为对照组,测量转基因线虫的寿命和产卵率.结果 成功获得hNgb转基因株系线虫,其寿命和产卵率与野生型N2和EGFP转基因线虫相比,差异无统计学意义.结论 外源性hNgb转基因不影响线虫的发育和正常生理功能,可用于Ngb功能的深入研究.%Objective To construct the transgenic Caenorhabditis elegans expressing human neuroglobin(hNgb) and to investigate the effect of hNgb on the brood size and lifespan of C. elegans. Methods The transgenic C. elegans expressing hNgb was obtained with microinjeetion. Expression of hNgb in the transgenic strains was detected by Western blot and immunohistochemistry. The life span and brood size of the C. elegans were detected using wild type N2, while the enhanced green fluorescin protein (EGFP) transgenic strain was used as a control. Results The hNgb transgenic strain was obtained. Compared with wild type C. elegans and EGFP transgenic strain, the transgenic hNgb C. elegans was no statistically significant difference in the life span and brood size. Conclusion The hNgb transgenic strain has no obvious effect on nematode's normal physiological function and could be used for the further functional identification of hNgb.

  16. Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

    DEFF Research Database (Denmark)

    Staunstrup, Nicklas Heine; Stenderup, Karin; Mortensen, Sidsel

    2017-01-01

    Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and tre...

  17. Increased fibrillar beta-amyloid in response to human clq injections into hippocampus and cortex of APP+PS1 transgenic mice.

    Science.gov (United States)

    Boyett, Kristal W; DiCarlo, Giovanni; Jantzen, Paul T; Jackson, Jennifer; O'Leary, Charlotte; Wilcock, Donna; Morgan, Dave; Gordon, Marcia N

    2003-01-01

    Human C1q when injected directly into hippocampus and cortex of doubly transgenic APP+PS1 mice results in the increase of Congo red-positive fibrillar deposits. Although there was no significant change in overall area stained for Abeta total, qualitatively it appeared that there was less diffuse Abeta in C1q-treated mice versus vehicle. There was no apparent change in astroglial or microglial activation caused by injection of C1q with respect to vehicle injections. These effects of C1q were only found in 50% BUB/BnJ mice, a strain with higher serum complement activity than other mouse lines. These in vivo data were consistent with the effects of C1q to increase fibrillogenesis of Abeta in vitro. In conclusion, complement protein C1q, believed to be involved in the pathogenesis of Alzheimer's disease in humans, can cause increased fibrillogenesis in the APP+PS1 mouse model of amyloid deposition.

  18. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.

    Science.gov (United States)

    Ban, Hiroshi; Nishishita, Naoki; Fusaki, Noemi; Tabata, Toshiaki; Saeki, Koichi; Shikamura, Masayuki; Takada, Nozomi; Inoue, Makoto; Hasegawa, Mamoru; Kawamata, Shin; Nishikawa, Shin-Ichi

    2011-08-23

    After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.

  19. Gender-specific effects on food intake but no inhibition of age-related fat accretion in transgenic mice overexpressing human IGFBP-2 lacking the Cardin-Weintraub sequence motif.

    Science.gov (United States)

    Wiedmer, Petra; Schwarz, Franziska; Große, Birgit; Schindler, Nancy; Tuchscherer, Armin; Russo, Vincenzo C; Tschöp, Matthias H; Hoeflich, Andreas

    2015-06-01

    IGFBP-2 affects growth and metabolism and is thought to impact on energy homeostasis and the accretion of body fat via its heparin binding domains (HBD). In order to assess the function of the HBD present in the linker domain (HBD1) we have generated transgenic mice overexpressing mutant human IGFBP-2 lacking the PKKLRP sequence and carrying a PNNLAP sequence instead. Transgenic mice expressed high amounts of human IGFBP-2, while endogenous IGFBP-2 or IGF-I serum concentrations were not affected. In both genders we performed a longitudinal analysis of growth and metabolism including at least 4 separate time points between the age of 10 and 52 weeks. Body composition was assessed by nuclear magnetic resonance (NMR) analysis. Food intake was recorded by an automated online-monitoring. We describe negative effects of mutant human IGFBP-2 on body weight, longitudinal growth and lean body mass (p transgenic mice of both genders (p transgene expression significantly increased absolute mass of total body fat over all age groups (p Food intake was increased in female but decreased in male transgenic mice at an age of 11 weeks. Thus our study clearly provides gender- and time-specific effects of HBD1-deficient hIGFBP-2 (H1d-BP-2) on fat mass accretion and food intake. While our data are in principal agreement with current knowledge on the role of HB-domains for fat accretion we now may also speculate on a role of HBD1 for the control of eating behavior.

  20. Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice.

    Science.gov (United States)

    Yueh, Mei-Fei; Tukey, Robert H

    2007-03-23

    The formation of beta-D-glucopyranosides (glucuronides) by the UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that facilitates the elimination of small hydrophobic molecules such as drugs, dietary constituents, steroids, and bile acids. We elucidate here that an anti-oxidative response leads to induction of UGT1A1 through the Nrf2-Keap1 pathway. When human HepG2 cells were treated with the prooxidants tert-butylhydroquinone and beta-naphthoflavone, cellular UGT1A1 glucuronidation activities were increased. The induction of UGT1A1 proceeded following the overexpression of Nrf2 and was blocked following overexpression of Keap1, demonstrating that Keap1 suppresses Nrf2 activation of the UGT1A1 gene. Loss of function analysis for Nrf2 conducted by small interfering RNA revealed that induction of UGT1A1 was not seen in Nrf2 knock-out cells. To examine the contribution of oxidants toward the regulation of human UGT1A1 in vivo, transgenic mice bearing the human UGT1 locus (Tg-UGT1) were treated with tert-butylhydroquinone. Human UGT1A1 was markedly increased in small and large intestines as well as in liver. Gene mapping experiments including transfections of UGT1A1 reporter gene constructs into HepG2 cells coupled with functional analysis of Nrf2 expression and binding to anti-oxidant-response elements (ARE) resulted in identification of an ARE in the phenobarbital-response enhancer module region of the UGT1A1 gene. The ARE flanks the recently identified Ah receptor xenobiotic-responsive element. The results suggest that Nrf2-Keap1-dependent UGT1A1 induction by prooxidants might represent a key adaptive response to cellular oxidative stress that defends against a variety of environmental insults, including electrophile attacks and chemical carcinogenesis.

  1. Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

    Institute of Scientific and Technical Information of China (English)

    Jian Zhu-Ge; Ying-Nian Yu; Yu-Li Qian; Xin Li

    2002-01-01

    AIM: The human cytochrome P-450 2C18(CYP2C18) hasbeen characterized. However, the protein has not beenpurified from liver and very little is known regarding thespecific substrate of CYP2C18. In order to study its enzymaticactivity for drug metabolism, the CYP2C18cDNA was clonedand a stable CHL cell line expressing recombinant CYP 2C18was established.METHODS: The human CYP2C18cDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector. The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREP9. A transgenic cell line was established bytransfecting the recombinant plasmid of pREPg-CYP2C18toChinese hamster lung (CHL) cell. The enzyme activity ofCYP2C18 catalyzing oxidation of tolbutamide tohydroxytolbutamide in postmitochondrial supernant(Sg)fraction of the cell was determined by high performanceliquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from thecloned cDNA segment was identical to that of reported byRomkes et al(GenBank accession number: M61856,J05326).The S9 fraction of the established cell line metabolizestolbutamide to hydroxytolbutamide. Tolbutamide hydroxylaseactivity was found to be 0.509±0.052 μmol.min-1.g-1 S9protein or 8.82±0.90 mol.min-1.mol-1 CYP, but wasundetectable in parental CHL cell. In addition, we haveidentified a CYP2C18cDNA clone with exon 5 missing.CONCLUSION: The cDNA of human CYP2C18 wassuccessfully cloned and a cell line, CHL-CYP2C18, efficientlyexpressing the protein of CYP2C18, was established. Aspliced variant of CYP2C18 with exon 5 missing was identifiedin the cloning process.

  2. Generation of red fluorescent protein transgenic dogs.

    Science.gov (United States)

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.

  3. Human herpesvirus 6A infection in CD46 transgenic mice: viral persistence in the brain and increased production of proinflammatory chemokines via Toll-like receptor 9.

    Science.gov (United States)

    Reynaud, Joséphine M; Jégou, Jean-François; Welsch, Jérémy C; Horvat, Branka

    2014-05-01

    Human herpesvirus 6 (HHV-6) is widely spread in the human population and has been associated with several neuroinflammatory diseases, including multiple sclerosis. To develop a small-animal model of HHV-6 infection, we analyzed the susceptibility of several lines of transgenic mice expressing human CD46, identified as a receptor for HHV-6. We showed that HHV-6A (GS) infection results in the expression of viral transcripts in primary brain glial cultures from CD46-expressing mice, while HHV-6B (Z29) infection was inefficient. HHV-6A DNA persisted for up to 9 months in the brain of CD46-expressing mice but not in the nontransgenic littermates, whereas HHV-6B DNA levels decreased rapidly after infection in all mice. Persistence in the brain was observed with infectious but not heat-inactivated HHV-6A. Immunohistological studies revealed the presence of infiltrating lymphocytes in periventricular areas of the brain of HHV-6A-infected mice. Furthermore, HHV-6A stimulated the production of a panel of proinflammatory chemokines in primary brain glial cultures, including CCL2, CCL5, and CXCL10, and induced the expression of CCL5 in the brains of HHV-6A-infected mice. HHV-6A-induced production of chemokines in the primary glial cultures was dependent on the stimulation of toll-like receptor 9 (TLR9). Finally, HHV-6A induced signaling through human TLR9 as well, extending observations from the murine model to human infection. Altogether, this study presents a first murine model for HHV-6A-induced brain infection and suggests a role for TLR9 in the HHV-6A-initiated production of proinflammatory chemokines in the brain, opening novel perspectives for the study of virus-associated neuropathology. HHV-6 infection has been related to neuroinflammatory diseases; however, the lack of a suitable small-animal infection model has considerably hampered further studies of HHV-6-induced neuropathogenesis. In this study, we have characterized a new model for HHV-6 infection in mice

  4. Comparison of Sirtuin 3 Levels in ALS and Huntington’s Disease—Differential Effects in Human Tissue Samples vs. Transgenic Mouse Models

    Directory of Open Access Journals (Sweden)

    Eva Buck

    2017-05-01

    Full Text Available Neurodegenerative diseases are characterized by distinct patterns of neuronal loss. In amyotrophic lateral sclerosis (ALS upper and lower motoneurons degenerate whereas in Huntington’s disease (HD medium spiny neurons in the striatum are preferentially affected. Despite these differences the pathophysiological mechanisms and risk factors are remarkably similar. In addition, non-neuronal features, such as weight loss implicate a dysregulation in energy metabolism. Mammalian sirtuins, especially the mitochondrial NAD+ dependent sirtuin 3 (SIRT3, regulate mitochondrial function and aging processes. SIRT3 expression depends on the activity of the metabolic master regulator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α, a modifier of ALS and HD in patients and model organisms. This prompted us to systematically probe Sirt3 mRNA and protein levels in mouse models of ALS and HD and to correlate these with patient tissue levels. We found a selective reduction of Sirt3 mRNA levels and function in the cervical spinal cord of end-stage ALS mice (superoxide dismutase 1, SOD1G93A. In sharp contrast, a tendency to increased Sirt3 mRNA levels was found in the striatum in HD mice (R6/2. Cultured primary neurons express the highest levels of Sirt3 mRNA. In primary cells from PGC-1α knock-out (KO mice the Sirt3 mRNA levels were highest in astrocytes. In human post mortem tissue increased mRNA and protein levels of Sirt3 were found in the spinal cord in ALS, while Sirt3 levels were unchanged in the human HD striatum. Based on these findings we conclude that SIRT3 mediates the different effects of PGC-1α during the course of transgenic (tg ALS and HD and in the human conditions only partial aspects Sirt3 dysregulation manifest.

  5. Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

    Directory of Open Access Journals (Sweden)

    Thorsen Jim

    2009-10-01

    Full Text Available Abstract Background Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency. Results We have constructed a shuttle vector, pPAC7, which contains both the EBNA-1 gene and oriP from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the EGFP reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the Bsr-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the CDH3 gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size. Conclusion The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes.

  6. Human cyclin T1 expression ameliorates a T-cell-specific transcriptional limitation for HIV in transgenic rats, but is not sufficient for a spreading infection of prototypic R5 HIV-1 strains ex vivo

    Directory of Open Access Journals (Sweden)

    Littman Dan R

    2009-01-01

    Full Text Available Abstract Background Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1. Results Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. Conclusion Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.

  7. High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs.

    Science.gov (United States)

    Lu, Dan; Li, Qiuyan; Wu, Zhibin; Shang, Shengzhe; Liu, Shen; Wen, Xiao; Li, Zhiyuan; Wu, Fangfang; Li, Ning

    2014-01-01

    Lysozyme is often used as a feed additive and acts as an antimicrobial protein that enhances immune function and defends against pathogenic bacteria in pigs. In this study, we genetically added recombinant human lysozyme (rhLZ) to sow milk by somatic cell nuclear transfer and investigated whether the presence of recombinant human lysozyme can influence intestinal microbiota and morphology in sucking pigs. We generated transgenic cloned pigs and the first-generation hybrids (F1) produced high levels of rhLZ in milk. The average concentration of rhLZ was 116.34 ± 24.46 mg/L in the milk of F1 sows, which was 1500-fold higher than that of the native pig lysozyme. In vitro, it was demonstrated that rhLZ in milk of transgenic pigs had enzyme levels at 92,272 ± 26,413 U/mL. In a feeding experiment, a total of 40 newborn piglets were nursed by four transgenic sows and four sibling non-transgenic sows (F1), with five piglets per gilt. The piglets were allowed to nurse for 21 days and the sow milk was the only source of nutrition for the piglets. All piglets were slaughtered on postnatal day 22. Six types of bacteria were cultured and analyzed to detect the impact of rhLZ on gut microbiota. The number of Escherichia coli in the duodenum of piglets reared by transgenic sows was significantly decreased (ptransgenic cloned pigs and elevated lysozyme level in nuring piglets. The results of the feeding experiments demonstrated that rhLZ-enhanced milk can inhibit the growth of E. coli in the duodenum and positively influence intestinal morphology without adversely affecting weight gain or piglet growth.

  8. Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

    Institute of Scientific and Technical Information of China (English)

    Bo WU; Yong Hua SUN; Yan Wu WANG; Ya Ping WANG; Zuo Yan ZHU

    2005-01-01

    The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-totail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class Ⅰ, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp β-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

  9. No renal phenotype in human phospholipid transfer protein transgenic apolipoprotein E deficient mice despite severe aortic atherosclerosis

    NARCIS (Netherlands)

    Dullaart, Robin P. F.; van Haperen, Rien; van den Born, Jaap; van Goor, Harry; de Crom, Rini; van Tol, Arie

    2014-01-01

    Background: Phospholipid transfer protein (PLTP) is an emerging cardiometabolic risk factor. Plasma PLTP is elevated in humans with end-stage kidney disease and glomerular proteinuria, but the contribution of systemic PLTP elevation to the development of renal damage is unknown. We tested whether hu

  10. Distribution of human umbilical cord blood-derived mesenchymal stem cells in the Alzheimer's disease transgenic mouse after a single intravenous injection.

    Science.gov (United States)

    Park, Sang Eon; Lee, Na Kyung; Lee, Jeongmin; Hwang, Jung Won; Choi, Soo Jin; Hwang, Hyeri; Hyung, Brian; Chang, Jong Wook; Na, Duk L

    2016-03-02

    The aim of this study was to track the migration of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) administered through a single intravenous injection and to observe the consequential therapeutic effects in a transgenic Alzheimer's disease mouse model. Ten-month-old APP/PS1 mice received a total injection of 1×10 cells through the lateral tail vein and were killed 1, 4, and 7 days after administration. On the basis of immunohistochemical analysis, hUCB-MSCs were not detected in the brain at any of the time points. Instead, most of the injected mesenchymal stem cells were found to be distributed in the lung, heart, and liver. In terms of the molecular effects, statistically significant differences in the amyloid β protein, neprilysin, and SOX2 levels were not observed among the groups. On the basis of the results from this study, we suggest that single intravenously administered hUCB-MSCs are not delivered to the brain and also do not have a significant influence on Alzheimer's disease pathology.

  11. Hippocampal changes produced by overexpression of the human CHRNA5/A3/B4 gene cluster may underlie cognitive deficits rescued by nicotine in transgenic mice.

    Science.gov (United States)

    Molas, Susanna; Gener, Thomas; Güell, Jofre; Martín, Mairena; Ballesteros-Yáñez, Inmaculada; Sanchez-Vives, Maria V; Dierssen, Mara

    2014-11-11

    Addiction involves long-lasting maladaptive changes including development of disruptive drug-stimuli associations. Nicotine-induced neuroplasticity underlies the development of tobacco addiction but also, in regions such as the hippocampus, the ability of this drug to enhance cognitive capabilities. Here, we propose that the genetic locus of susceptibility to nicotine addiction, the CHRNA5/A3/B4 gene cluster, encoding the α5, α3 and β4 subunits of the nicotinic acetylcholine receptors (nAChRs), may influence nicotine-induced neuroadaptations. We have used transgenic mice overexpressing the human cluster (TgCHRNA5/A3/B4) to investigate hippocampal structure and function in genetically susceptible individuals. TgCHRNA5/A3/B4 mice presented a marked reduction in the dendrite complexity of CA1 hippocampal pyramidal neurons along with an increased dendritic spine density. In addition, TgCHRNA5/A3/B4 exhibited increased VGLUT1/VGAT ratio in the CA1 region, suggesting an excitatory/inhibitory imbalance. These hippocampal alterations were accompanied by a significant impairment in short-term novelty recognition memory. Interestingly, chronic infusion of nicotine (3.25 mg/kg/d for 7 d) was able to rescue the reduced dendritic complexity, the excitatory/inhibitory imbalance and the cognitive impairment in TgCHRNA5/A3/B4. Our results suggest that chronic nicotine treatment may represent a compensatory strategy in individuals with altered expression of the CHRNA5/A3/B4 region.

  12. Targeted overexpression of the human urotensin receptor transgene in smooth muscle cells: effect of UT antagonism in ApoE knockout mice fed with Western diet.

    Science.gov (United States)

    Papadopoulos, Panayiota; Bousette, Nicolas; Al-Ramli, Wisam; You, Zhipeng; Behm, David J; Ohlstein, Eliot H; Harrison, Stephen M; Douglas, Stephen A; Giaid, Adel

    2009-06-01

    Urotensin II (UII) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study we used four strains of mice; wild-type (WT), UT(+) (a transgenic strain expressing human UT driven by the alpha-smooth muscle-specific, SM22, promoter), ApoE knockout (ko), and UT(+)/ApoE ko. All animals were fed high fat diet for 12 weeks. Western blot analysis revealed a significant increase in aortic UT expression in UT(+) relative to WT mice (PUT protein level to that of UT(+). Immunohistochemistry revealed the presence of strong expression of UT and UII proteins in the atheroma of UT(+), ApoE ko and UT(+)/ApoE ko mice, particularly in foam cells. Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT(+)/ApoE ko but not in UT(+) mice when compared to WT mice (PUT(+), ApoE ko and UT(+)/ApoE ko compared to WT mice (PUT receptor antagonist SB-657510A (30 microg/Kg/day gavage) for 10 weeks in a group of ApoE ko mice fed on high fat diet resulted in a significant reduction of lesion (PUT in the pathogenesis of atherosclerosis. The use of UT receptor antagonists may provide a beneficial tool in the management of this debilitating disease process.

  13. Intracerebroventricular transplantation of human amniotic epithelial cells ameliorates spatial memory deficit in the doubly transgenic mice coexpressing APPswe and PS1ΔE9-deleted genes

    Institute of Scientific and Technical Information of China (English)

    XUE Shou-ru; CHEN Chong-fang; DONG Wan-li; HUI Guo-zhen; LIU Tian-jun; GUO Li-he

    2011-01-01

    Background Human amniotic epithelial cells (HAECs),which have characteristics of both embryonic and pluripotent stem cells,are therefore a candidate in cell therapy without creating legal or ethical problems.In the present study,we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer's disease (AD) coexpressing presenilin-1 (PS1) and mutant Sweden amyloid precursor protein (APPswe)genes.Methods The offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene.The doubly transgenic (TG) mice (n=20) and wild-type (WT) mice (n=20) were randomly divided into two groups respectively:the transplantation group treated with HAECs and the control group with phosphate buffered saline.Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice.Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively.Immunofluorescence cytochemistry was used to track the survival of HAECs.Immunohistochemistry was used to determine the expression of octamer-binding protein 4 (Oct-4) and Nanog in the HAECs.High performance liquid chromatography was used to measure acetylcholine in hippocampus.The density of cholinergic neurons in basal forebrain and nerve fibers in hippocampus was measured using acetylcholinesterase staining.Results Amyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months,but not in WT mice.The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection.The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell.Compared with the control group,transplantation of HAECs can not only significantly improve the spatial memory of the TG mice,but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites.Conclusions These

  14. Transgenic animals and their application in medicine

    Directory of Open Access Journals (Sweden)

    Bagle TR, Kunkulol RR, Baig MS, More SY

    2013-01-01

    Full Text Available Transgenic animals are animals that are genetically altered to have traits that mimic symptoms of specific human pathologies. They provide genetic models of various human diseases which are important in understanding disease and developing new targets. In early 1980 Gordon and co-workers described the first gene addition experiment using the microinjection technology and since then the impact of transgenic technology on basic research has been significant. Within 20 years of its inception, ATryn the first drug approved by USFDA from transgenic animals was developed and it has opened door to drugs from transgenic animals. In addition, they are looked upon as potential future donors for xenotransplantation. With increasing knowledge about the genetics and improvements in the transgenetic technology numerous useful applications like biologically safe new-generation drugs based on human regulatory proteins are being developed.Various aspects of concern in the coming years are the regulatory guidelines, ethical issues and patents related to the use of transgenic animals. This modern medicine is on the threshold of a pharmacological revolution. Use of transgenic animals will provide solutions for drug research, xenotransplantation, clinical trials and will prove to be a new insight in drug development.

  15. Deletion of the core region of 5' HS2 of the mouse beta-globin locus control region reveals a distinct effect in comparison with human beta-globin transgenes.

    Science.gov (United States)

    Hu, Xiao; Bulger, Michael; Bender, M A; Fields, Jennifer; Groudine, Mark; Fiering, Steven

    2006-01-15

    The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp "flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However, human transgenes in which only individual HS core regions were deleted showed drastic loss of expression accompanied by changes in chromatin structure. To address these disparate results, we have deleted the core region of 5'HS2 from the endogenous murine beta-LCR. The phenotype was similar to that of the larger 5'HS2 deletion, with no apparent disruption of chromatin structure. These results demonstrate that the greater severity of HS core deletions in comparison to full HS deletions is not a general property of the beta-LCR.

  16. Deletion of the core region of 5′ HS2 of the mouse β-globin locus control region reveals a distinct effect in comparison with human β-globin transgenes

    Science.gov (United States)

    Hu, Xiao; Bulger, Michael; Bender, M. A.; Fields, Jennifer; Groudine, Mark; Fiering, Steven

    2006-01-01

    The β-globin locus control region (LCR) is a large DNA element that is required for high-level expression of β-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human β-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp “flanks” with weaker functional activity and lower interspecies homology. Studies of human β-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However, human transgenes in which only individual HS core regions were deleted showed drastic loss of expression accompanied by changes in chromatin structure. To address these disparate results, we have deleted the core region of 5′HS2 from the endogenous murine β-LCR. The phenotype was similar to that of the larger 5′HS2 deletion, with no apparent disruption of chromatin structure. These results demonstrate that the greater severity of HS core deletions in comparison to full HS deletions is not a general property of the β-LCR. (Blood. 2006;107:821-826) PMID:16189270

  17. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.

    OpenAIRE

    Rath, H. C.; Herfarth, H H; Ikeda, J S; Grenther, W B; Hamm, T. E.; Balish, E.; Taurog, J D; Hammer, R. E.; Wilson, K. H.; Sartor, R B

    1996-01-01

    Genetic and environmental factors are important in the pathogenesis of clinical and experimental chronic intestinal inflammation. We investigated the influence of normal luminal bacteria and several groups of selected bacterial strains on spontaneous gastrointestinal and systemic inflammation in HLA-B27 transgenic rats. Rats maintained germfree for 3-9 mo were compared with littermates conventionalized with specific pathogen-free bacteria. Subsequently, germfree transgenic rats were colonized...

  18. Sheep and Goat BSE Propagate More Efficiently than Cattle BSE in Human PrP Transgenic Mice

    OpenAIRE

    2011-01-01

    A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work ...

  19. Generation of BAC transgenic epithelial organoids.

    Directory of Open Access Journals (Sweden)

    Gerald Schwank

    Full Text Available Under previously developed culture conditions, mouse and human intestinal epithelia can be cultured and expanded over long periods. These so-called organoids recapitulate the three-dimensional architecture of the gut epithelium, and consist of all major intestinal cell types. One key advantage of these ex vivo cultures is their accessibility to live imaging. So far the establishment of transgenic fluorescent reporter organoids has required the generation of transgenic mice, a laborious and time-consuming process, which cannot be extended to human cultures. Here we present a transfection protocol that enables the generation of recombinant mouse and human reporter organoids using BAC (bacterial artificial chromosome technology.

  20. Screening of human tumor antigens for CD4 T cell epitopes by combination of HLA-transgenic mice, recombinant adenovirus and antigen peptide libraries.

    Directory of Open Access Journals (Sweden)

    Wolfram Osen

    Full Text Available BACKGROUND: As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. METHODS AND FINDINGS: To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1 or TRP-2 (Ad5.TRP-2 were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2(60-74 epitope and against the new epitope TRP-2(149-163. Importantly, human T cells specifically recognizing target cells loaded with the TRP-2(149-163-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1(284-298 as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. CONCLUSIONS: Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target

  1. Pharming and transgenic plants.

    Science.gov (United States)

    Liénard, David; Sourrouille, Christophe; Gomord, Véronique; Faye, Loïc

    2007-01-01

    Plant represented the essence of pharmacopoeia until the beginning of the 19th century when plant-derived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. In the last decades, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. More recently, molecular farming has rapidly pushed towards plants among the major players in recombinant protein production systems. Indeed, therapeutic protein production is safe and extremely cost-effective in plants. Unlike microbial fermentation, plants are capable of carrying out post-translational modifications and, unlike production systems based on mammalian cell cultures, plants are devoid of human infective viruses and prions. Furthermore, a large panel of strategies and new plant expression systems are currently developed to improve the plant-made pharmaceutical's yields and quality. Recent advances in the control of post-translational maturations in transgenic plants will allow them, in the near future, to perform human-like maturations on recombinant proteins and, hence, make plant expression systems suitable alternatives to animal cell factories.

  2. Studies of an expanded trinucleotide repeat in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Bingham, P.; Wang, S.; Merry, D. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    1994-09-01

    Spinal and bulbar muscular atrophy (SBMA) is a progressive motor neuron disease caused by expansion of a trinucleotide repeat in the androgen receptor gene (AR{sup exp}). AR{sup exp} repeats expand further or contract in approximately 25% of transmissions. Analogous {open_quotes}dynamic mutations{close_quotes} have been reported in other expanded trinucleotide repeat disorders. We have been developing a mouse model of this disease using a transgenic approach. Expression of the SBMA AR was documented in transgenic mice with an inducible promoter. No phenotypic effects of transgene expression were observed. We have extended our previous results on stability of the expanded trinucleotide repeat in transgenic mice in two lines carrying AR{sup exp}. Tail DNA was amplified by PCR using primers spanning the repeat on 60 AR{sup exp} transgenic mice from four different transgenic lines. Migration of the PCR product through an acrylamide gel showed no change of the 45 CAG repeat length in any progeny. Similarly, PCR products from 23 normal repeat transgenics showed no change from the repeat length of the original construct. Unlike the disease allele in humans, the expanded repeat AR cDNA in transgenic mice showed no change in repeat length with transmission. The relative stability of CAG repeats seen in the transgenic mice may indicate either differences in the fidelity of replicative enzymes, or differences in error identification and repair between mice and humans. Integration site or structural properties of the transgene itself might also play a role.

  3. Embryonic catalase protects against ethanol-initiated DNA oxidation and teratogenesis in acatalasemic and transgenic human catalase-expressing mice.

    Science.gov (United States)

    Miller, Lutfiya; Shapiro, Aaron M; Wells, Peter G

    2013-08-01

    Reactive oxygen species (ROS) are implicated in fetal alcohol spectrum disorders (FASD) caused by alcohol (ethanol, EtOH). Although catalase detoxifies hydrogen peroxide, embryonic catalase activity is only about 5% of maternal levels. To determine the roles of ROS and embryonic catalase in FASD, pregnant mice with enhanced (expressing human catalase, hCat) or deficient (acatalasemic, aCat) catalase activity, or their respective wild-type (WT) controls, were treated ip on gestational day 9 with 4 or 6g/kg EtOH or its saline vehicle, and embryos and fetuses were, respectively, evaluated for oxidatively damaged DNA and structural anomalies. Untreated hCat and aCat dams had, respectively, more and less offspring than their WT controls. hCat progenies were protected from all EtOH fetal anomalies at the low dose (p fetal anomalies (p fetal anomalies (p teratogenesis. Endogenous embryonic catalase, despite its low level, is an important embryoprotective enzyme for EtOH teratogenesis and a likely determinant of individual risk.

  4. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

    Science.gov (United States)

    Miller-Pinsler, Lutfiya; Wells, Peter G

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (pcatalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (pcatalase is a determinant of risk for EtOH embryopathies.

  5. Ebola Virus Replication and Disease Without Immunopathology in Mice Expressing Transgenes to Support Human Myeloid and Lymphoid Cell Engraftment.

    Science.gov (United States)

    Spengler, Jessica R; Lavender, Kerry J; Martellaro, Cynthia; Carmody, Aaron; Kurth, Andreas; Keck, James G; Saturday, Greg; Scott, Dana P; Nichol, Stuart T; Hasenkrug, Kim J; Spiropoulou, Christina F; Feldmann, Heinz; Prescott, Joseph

    2016-10-15

    The study of Ebola virus (EBOV) pathogenesis in vivo has been limited to nonhuman primate models or use of an adapted virus to cause disease in rodent models. Herein we describe wild-type EBOV (Makona variant) infection of mice engrafted with human hematopoietic CD34(+) stem cells (Hu-NSG™-SGM3 mice; hereafter referred to as SGM3 HuMice). SGM3 HuMice support increased development of myeloid immune cells, which are primary EBOV targets. In SGM3 HuMice, EBOV replicated to high levels, and disease was observed following either intraperitoneal or intramuscular inoculation. Despite the high levels of viral antigen and inflammatory cell infiltration in the liver, the characteristic histopathology of Ebola virus disease was not observed, and this absence of severe immunopathology may have contributed to the recovery and survival of some of the animals. Future investigations into the underlying mechanisms of the atypical disease presentation in SGM3 HuMice will provide additional insights into the immunopathogenesis of severe EBOV disease. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  6. Development of a humanized HLA-A2.1/DP4 transgenic mouse model and the use of this model to map HLA-DP4-restricted epitopes of HBV envelope protein.

    Directory of Open Access Journals (Sweden)

    Zhitao Ru

    Full Text Available A new homozygous humanized transgenic mouse strain, HLA-A2.1(+/+HLA-DP4(+/+ hCD4(+/+mCD4(-/-IAβ(-/-β2m(-/- (HLA-A2/DP4, was obtained by crossing the previously characterized HLA-A2(+/+β2m(-/- (A2 mouse and our previously created HLA-DP4(+/+ hCD4(+/+mCD4(-/-IAβ(-/- (DP4 mouse. We confirmed that the transgenes (HLA-A2, HLA-DP4, hCD4 inherited from the parental A2 and DP4 mice are functional in the HLA-A2/DP4 mice. After immunizing HLA-A2/DP4 mice with a hepatitis B DNA vaccine, hepatitis B virus-specific antibodies, HLA-A2-restricted and HLA-DP4-restricted responses were observed to be similar to those in naturally infected humans. Therefore, the present study demonstrated that HLA-A2/DP4 transgenic mice can faithfully mimic human cellular responses. Furthermore, we reported four new HLA-DP4-restricted epitopes derived from HBsAg that were identified in both vaccinated HLA-A2/DP4 mice and HLA-DP4-positive human individuals. The HLA-A2/DP4 mouse model is a promising preclinical animal model carrying alleles present to more than a quarter of the human population. This model should facilitate the identification of novel HLA-A2- and HLA-DP4-restricted epitopes and vaccine development as well as the characterization of HLA-DP4-restricted responses against infection in humans.

  7. Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

    Science.gov (United States)

    Harel-Markowitz, Eliane; Gurevich, Michael; Shore, Laurence S; Katz, Adi; Stram, Yehuda; Shemesh, Mordechai

    2009-05-01

    Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

  8. High-level recombinant human lysozyme expressed in milk of transgenic pigs can inhibit the growth of Escherichia coli in the duodenum and influence intestinal morphology of sucking pigs.

    Directory of Open Access Journals (Sweden)

    Dan Lu

    Full Text Available Lysozyme is often used as a feed additive and acts as an antimicrobial protein that enhances immune function and defends against pathogenic bacteria in pigs. In this study, we genetically added recombinant human lysozyme (rhLZ to sow milk by somatic cell nuclear transfer and investigated whether the presence of recombinant human lysozyme can influence intestinal microbiota and morphology in sucking pigs. We generated transgenic cloned pigs and the first-generation hybrids (F1 produced high levels of rhLZ in milk. The average concentration of rhLZ was 116.34 ± 24.46 mg/L in the milk of F1 sows, which was 1500-fold higher than that of the native pig lysozyme. In vitro, it was demonstrated that rhLZ in milk of transgenic pigs had enzyme levels at 92,272 ± 26,413 U/mL. In a feeding experiment, a total of 40 newborn piglets were nursed by four transgenic sows and four sibling non-transgenic sows (F1, with five piglets per gilt. The piglets were allowed to nurse for 21 days and the sow milk was the only source of nutrition for the piglets. All piglets were slaughtered on postnatal day 22. Six types of bacteria were cultured and analyzed to detect the impact of rhLZ on gut microbiota. The number of Escherichia coli in the duodenum of piglets reared by transgenic sows was significantly decreased (p<0.001 and their villus height to crypt depth ratio in the intestine were increased due to the significant decrease of crypt depth in the duodenum, jejunum, and ileum (p<0.001. Together, we successfully generated rhLZ transgenic cloned pigs and elevated lysozyme level in nuring piglets. The results of the feeding experiments demonstrated that rhLZ-enhanced milk can inhibit the growth of E. coli in the duodenum and positively influence intestinal morphology without adversely affecting weight gain or piglet growth.

  9. Risks and concerns regarding transgenic food and human health Riesgos y preocupaciones sobre los alimentos transgénicos y la salud humana

    Directory of Open Access Journals (Sweden)

    Acosta Orlando

    2002-12-01

    Full Text Available The transgenic technology in agriculture has recently been in the center of an intense debate between two radically opposite points of view. Some non-government organizations (NGO consider this technology as dangerous for human health, environment and economics of developing countries. On the contrary, the scientific community has been publicly supportive of this technology, suggesting that education is the key to gaining the public acceptance. Although genetically modified (GM plants for food use might have the potential to provide benefits in food quality, nutrition, health and environment, GM plants need additional considerations related with biosafety. Despite there is not evidence that GM foods are more dangerous to human health than conventional food it is necessary to test GM food following the best scientific methodology available. This review focuses on the potential effects that GM food might have on human health. La tecnología transgénica en la agricultura se ha visto recientemente implicada en un intenso debate entre dos corrientes opuestas. Algunas organizaciones no gubernamentales (ONG consideran esta tecnología como de alto riesgo para la salud, el ambiente y la economía de los países no desarrollados. Por el contrario, la comunidad científica ha respaldado públicamente esta tecnología asegurando que la clave para su aceptación es la educación. Aunque los alimentos genéticamente modificados (GM poseen el potencial de proveer beneficios en calidad alimentaria, salud y ambiente, es necesario considerarlos dentro de estrictos protocolos de bioseguridad. No obstante la ausencia de evidencias acerca de que los alimentos transgénicos son más peligrosos para la salud que los alimentos convencionales, aquellos transgénicos deben ser examinados con las metodologías científicas más rigurosas disponibles. Esta revisión se centra en el efecto potencial de los alimentos transgénicos sobre la salud humana. 

  10. Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

    Science.gov (United States)

    Wu, Chia-Ying; Lin, Yi-Wen; Kuo, Chia-Ho; Liu, Wan-Hsin; Tai, Hsiu-Fen; Pan, Chien-Hung; Chen, Yung-Tsung; Hsiao, Pei-Wen; Chan, Chi-Hsien; Chang, Ching-Chuan; Liu, Chung-Cheng; Chow, Yen-Hung; Chen, Juine-Ruey

    2015-01-01

    Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10(7) TCID50/mL 10 days after infection when using an MOI of 10(-4). The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.

  11. Expression of tryptophan 2,3-dioxygenase and production of kynurenine pathway metabolites in triple transgenic mice and human Alzheimer's disease brain.

    Directory of Open Access Journals (Sweden)

    Wei Wu

    Full Text Available To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD, the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO, and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

  12. Glucose lowering effect of transgenic human insulin-like growth factor-I from rice: in vitro and in vivo studies

    Directory of Open Access Journals (Sweden)

    Sun Samuel SM

    2011-04-01

    Full Text Available Abstract Background Human insulin-like growth factor-I (hIGF-I is a growth factor which is highly resemble to insulin. It is essential for cell proliferation and has been proposed for treatment of various endocrine-associated diseases including growth hormone insensitivity syndrome and diabetes mellitus. In the present study, an efficient plant expression system was developed to produce biologically active recombinant hIGF-I (rhIGF-I in transgenic rice grains. Results The plant-codon-optimized hIGF-I was introduced into rice via Agrobacterium-mediated transformation. To enhance the stability and yield of rhIGF-I, the endoplasmic reticulum-retention signal and glutelin signal peptide were used to deliver rhIGF-I to endoplasmic reticulum for stable accumulation. We found that only glutelin signal peptide could lead to successful expression of hIGF-I and one gram of hIGF-I rice grain possessed the maximum activity level equivalent to 3.2 micro molar of commercial rhIGF-I. In vitro functional analysis showed that the rice-derived rhIGF-I was effective in inducing membrane ruffling and glucose uptake on rat skeletal muscle cells. Oral meal test with rice-containing rhIGF-I acutely reduced blood glucose levels in streptozotocin-induced and Zucker diabetic rats, whereas it had no effect in normal rats. Conclusion Our findings provided an alternative expression system to produce large quantities of biologically active rhIGF-I. The provision of large quantity of recombinant proteins will promote further research on the therapeutic potential of rhIGF-I.

  13. Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

    Science.gov (United States)

    Panahi, Mitra; Alli, Zaman; Cheng, Xiongying; Belbaraka, Loubaba; Belgoudi, Jaafar; Sardana, Ravinder; Phipps, Jenny; Altosaar, Illimar

    2004-06-01

    Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been

  14. Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Miller-Pinsler, Lutfiya [Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario (Canada); Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada)

    2015-09-15

    Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

  15. Production of recombinant proteins in milk of transgenic and non-transgenic goats

    Directory of Open Access Journals (Sweden)

    Raylene Ramos Moura

    2011-10-01

    Full Text Available Among all the transgenic mammalians produced so far, goats have represented an excellent model of transgenesis when considering the factors such as the market demand for protein, volume of milk produced per lactation and reproductive rate. Various recombinant proteins have been obtained from the transgenic and non-transgenic goats, and among these, human antithrombin, produced by the transgenic goats, was the first recombinant protein of animal origin to be released as a drug for the clinical use in humans. This review reports the aspects inherent to the production of recombinant proteins in the goats, from the production of the animal bioreactors up to the expression of these proteins in their milk.

  16. Pathologic progression of mammary carcinomas in a C3(1)/SV40 T/t-antigen transgenic rat model of human triple-negative and Her2-positive breast cancer.

    Science.gov (United States)

    Hoenerhoff, M J; Shibata, M A; Bode, A; Green, J E

    2011-04-01

    The C3(1) component of the rat prostate steroid binding protein has been used to target expression of the SV40 T/t-antigen to the mammary epithelium of mice resulting in pre-neoplastic lesions that progress to invasive and metastatic cancer with molecular features of human basal-type breast cancer. However, there are major differences in the histologic architecture of the stromal and epithelial elements between the mouse and human mammary glands. The rat mammary gland is more enriched with epithelial and stromal components than the mouse and more closely resembles the cellular composition of the human gland. Additionally, existing rat models of mammary cancer are typically estrogen receptor positive and hormone responsive, unlike most genetically engineered mouse mammary cancer models. In an attempt to develop a mammary cancer model that might more closely resemble the pathology of human breast cancer, we generated a novel C3(1)/SV40 T/t-antigen transgenic rat model that developed progressive mammary lesions leading to highly invasive adenocarcinomas. However, aggressive tumor development prevented the establishment of transgenic lines. Characterization of the tumors revealed that they were primarily estrogen receptor and progesterone receptor negative, and either her2/neu positive or negative, resembling human triple-negative or Her2 positive breast cancer. Tumors expressed the basal marker K14, as well as the luminal marker K18, and were negative for smooth muscle actin. The triple negative phenotype has not been previously reported in a rat mammary cancer model. Further development of a C3(1)SV40 T/t-antigen based model could establish valuable transgenic rat lines that develop basal-type mammary tumors.

  17. No dopamine cell loss or changes in cytoskeleton function in transgenic mice expressing physiological levels of wild type or G2019S mutant LRRK2 and in human fibroblasts.

    Science.gov (United States)

    Garcia-Miralles, Marta; Coomaraswamy, Janaky; Häbig, Karina; Herzig, Martin C; Funk, Natalja; Gillardon, Frank; Maisel, Martina; Jucker, Mathias; Gasser, Thomas; Galter, Dagmar; Biskup, Saskia

    2015-01-01

    Mutations within the LRRK2 gene have been identified in Parkinson's disease (PD) patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.

  18. No dopamine cell loss or changes in cytoskeleton function in transgenic mice expressing physiological levels of wild type or G2019S mutant LRRK2 and in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Marta Garcia-Miralles

    Full Text Available Mutations within the LRRK2 gene have been identified in Parkinson's disease (PD patients and have been implicated in the dysfunction of several cellular pathways. Here, we explore how pathogenic mutations and the inhibition of LRRK2 kinase activity affect cytoskeleton dynamics in mouse and human cell systems. We generated and characterized a novel transgenic mouse model expressing physiological levels of human wild type and G2019S-mutant LRRK2. No neuronal loss or neurodegeneration was detected in midbrain dopamine neurons at the age of 12 months. Postnatal hippocampal neurons derived from transgenic mice showed no alterations in the seven parameters examined concerning neurite outgrowth sampled automatically on several hundred neurons using high content imaging. Treatment with the kinase inhibitor LRRK2-IN-1 resulted in no significant changes in the neurite outgrowth. In human fibroblasts we analyzed whether pathogenic LRRK2 mutations change cytoskeleton functions such as cell adhesion. To this end we compared the adhesion characteristics of human skin fibroblasts derived from six PD patients carrying one of three different pathogenic LRRK2 mutations and from four age-matched control individuals. The mutant LRRK2 variants as well as the inhibition of LRRK2 kinase activity did not reveal any significant cell adhesion differences in cultured fibroblasts. In summary, our results in both human and mouse cell systems suggest that neither the expression of wild type or mutant LRRK2, nor the inhibition of LRRK2 kinase activity affect neurite complexity and cellular adhesion.

  19. Developments in transgenic technology: applications for medicine.

    Science.gov (United States)

    Hunter, Cheryl V; Tiley, Laurence S; Sang, Helen M

    2005-06-01

    Recent advances in the efficiency of transgenic technology have important implications for medicine. The production of therapeutic proteins from animal bioreactors is well established and the first products are close to market. The genetic modification of pigs to improve their suitability as organ donors for xenotransplantation has been initiated, but many challenges remain. The use of transgenesis, in combination with the method of RNA interference to knock down gene expression, has been proposed as a method for making animals resistant to viral diseases, which could reduce the likelihood of transmission to humans. Here, the latest developments in transgenic technology and their applications relevant to medicine and human health will be discussed.

  20. Short Fibrils Constitute the Major Species of Seed-Competent Tau in the Brains of Mice Transgenic for Human P301S Tau.

    Science.gov (United States)

    Jackson, Samuel J; Kerridge, Caroline; Cooper, Jane; Cavallini, Annalisa; Falcon, Benjamin; Cella, Claire V; Landi, Alessia; Szekeres, Philip G; Murray, Tracey K; Ahmed, Zeshan; Goedert, Michel; Hutton, Michael; O'Neill, Michael J; Bose, Suchira

    2016-01-20

    The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. Significance statement: The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimer's disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein

  1. Relationship between ubiquilin-1 and BACE1 in human Alzheimer's disease and APdE9 transgenic mouse brain and cell-based models.

    Science.gov (United States)

    Natunen, Teemu; Takalo, Mari; Kemppainen, Susanna; Leskelä, Stina; Marttinen, Mikael; Kurkinen, Kaisa M A; Pursiheimo, Juha-Pekka; Sarajärvi, Timo; Viswanathan, Jayashree; Gabbouj, Sami; Solje, Eino; Tahvanainen, Eveliina; Pirttimäki, Tiina; Kurki, Mitja; Paananen, Jussi; Rauramaa, Tuomas; Miettinen, Pasi; Mäkinen, Petra; Leinonen, Ville; Soininen, Hilkka; Airenne, Kari; Tanzi, Rudolph E; Tanila, Heikki; Haapasalo, Annakaisa; Hiltunen, Mikko

    2016-01-01

    Accumulation of β-amyloid (Aβ) and phosphorylated tau in the brain are central events underlying Alzheimer's disease (AD) pathogenesis. Aβ is generated from amyloid precursor protein (APP) by β-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aβ pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aβ40 and Aβ42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co

  2. 转人α-乳清白蛋白基因奶粉亚慢性毒性试验%Study on sub-chronic toxicity of powered milk containing transgenic human α-lactalbumin

    Institute of Scientific and Technical Information of China (English)

    支媛; 徐海滨; 刘海波; 耿桂英; 汪会玲; 杨华; 冯晓莲; 高芃; 余强; 冯永全

    2011-01-01

    Objective To investigate the potential toxic or adverse effect of transgenic human α-lactalbumin powered milk on rats.Methods Weanling Wistar rats were randomly divided into seven groups according the weight: three transgenic milk powder(T) groups, three non-transgenic milk powder(N) groups and the control(C)group.The diets of T groups contain 15% , 30% and 60% transgenic human α-lactalbumin milk powder.The diets of N groups contain 15% ,30% and 60% non-transgenic human α-lactalbumin milk powder for 90 days.The diet of C group contains only basic feed.Haematological and biochemical parameters was measured during the study ( at 45th and 90th of the experiment).At the end of the 90th day, organ tissues analysis was performed.Results There were no transgenic human α-lactalbumin related adverse effects on the body weight, food intake, food consumption,hematology,serum biochemistry, as well as histopathology.Conclusion There were no signs of toxic and adverse effects for transgenic human α-lactalbumin powdered milk on rats.%目的 研究转人α-乳清白蛋白基因奶粉对Wistar大鼠的亚慢性毒性.方法 取初断乳Wistar大鼠140只,雌雄各半,按照体重随机分为7组.分别喂饲含有转人α-乳清白蛋白比例为15%、30%和60%成分奶粉的配制饲料,添加比例为15%、30%和60%普通奶粉的配制饲料和基础饲料,喂饲90天.在实验中期(第45天)和实验结束时(第90天)采血检测血液学和血液生化指标.实验结束时处死实验动物,测定脏器绝对重量,计算脏器指数,并对主要脏器进行病理组织学观察.结果 转人α-乳清白蛋白基因奶粉组与普通奶粉组和基础饲料组比较,未发现转人α-乳清白蛋白基因奶粉对动物体重、食物利用率、血液学、血液生化、脏器系数和病理组织学观察有生物学意义的改变.结论 给予大鼠转人α-乳清白蛋白基因奶粉90天未发现该奶粉对实验动物有毒性作用.

  3. Transgene Stacking in Cotton Improvement

    Institute of Scientific and Technical Information of China (English)

    YANG Ye-hua; WANG Xue-kui; YAO Ming-jing; FAN Yu-peng; GAO Da-yu

    2008-01-01

    @@ To date,more and more transgenic varieties of upland cotton (Gossypium hirsuturn L.) generated with transgenes,which derived from varies of alien species,are playing important role in agricultural production.Stacking of multi-transgenes has a potential for combining all the merits of distinct transgenic lines in a cultivar and possibly makes a significant contribution to cultivar improvement.

  4. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  5. Weeding with transgenes.

    Science.gov (United States)

    Duke, Stephen O

    2003-05-01

    Transgenes promise to reduce insecticide and fungicide use but relatively little has been done to significantly reduce herbicide use through genetic engineering. Recently, three strategies for transgene utilization have been developed that have the potential to change this. These are the improvement of weed-specific biocontrol agents, enhancement of crop competition or allelopathic traits, and production of cover crops that will self-destruct near the time of planting. Failsafe risk mitigation technologies are needed for most of these strategies.

  6. Human p53(264-272) HLA-A2 binding peptide is an immunodominant epitope in DNA-immunized HLA-A2 transgenic mice

    DEFF Research Database (Denmark)

    Petersen, T R; Bregenholta, S; Pedersen, L O;

    1999-01-01

    C57BL/10 mice transgenic for HLA-A2 were immunized with either a full-length DNA-construct of the tumor suppressor p53 or with a minigene encoding the p53-derived immunodominant peptide p53(264)LLGRNSFEV272 (L9V). Vaccination with the full-length p53 construct induced potent cytotoxic activity...

  7. Establishment of a retinoic acid-resistant human acute promyelocytic leukaemia (APL) model in human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic severe combined immunodeficiency (SCID) mice.

    Science.gov (United States)

    Fukuchi, Y; Kizaki, M; Kinjo, K; Awaya, N; Muto, A; Ito, M; Kawai, Y; Umezawa, A; Hata, J; Ueyama, Y; Ikeda, Y

    1998-10-01

    To understand the mechanisms and identify novel approaches to overcoming retinoic acid (RA) resistance in acute promyelocytic leukaemia (APL), we established the first human RA-resistant APL model in severe combined immunodeficiency (SCID) mice. UF-1 cells, an RA-resistant APL cell line established in our laboratory, were transplanted into human granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing SCID (hGMTg SCID) mice and inoculated cells formed subcutaneous tumours in all hGMTg SCID mice, but not in the non-transgenic control SCID mice. Single-cell suspensions (UF-1/GMTg SCID cells) were similar in morphological, immunological, cytogenetic and molecular genetic features to parental UF-1 cells. All-trans RA did not change the morphological features of cells or their expression of CD11b. RA did not alter the growth curve of cells as determined by MTT assay, suggesting that UF-1/GMTg SCID cells are resistant to RA. These results demonstrate that this is the first RA-resistant APL animal model that may be useful for investigating the biology of this myeloid leukaemia in vivo, as well as for evaluating novel therapeutic approaches including patients with RA-resistant APL.

  8. A human apoB100 transgenic mouse expresses human apoB100 in the RPE and develops features of early AMD

    DEFF Research Database (Denmark)

    Fujihara, Masashi; Bartels, Emil; Nielsen, Lars B

    2009-01-01

    changes consistent with early human AMD including loss of basal infoldings and accumulation of cytoplasmic vacuoles in the RPE, and basal laminar deposits containing long-spacing collagen and heterogeneous debris in Bruch membrane of apoB100 mice. In apoB100 mice given a high-fat diet, basal linear...

  9. Short-term suppression of A315T mutant human TDP-43 expression improves functional deficits in a novel inducible transgenic mouse model of FTLD-TDP and ALS.

    Science.gov (United States)

    Ke, Yazi D; van Hummel, Annika; Stevens, Claire H; Gladbach, Amadeus; Ippati, Stefania; Bi, Mian; Lee, Wei S; Krüger, Sarah; van der Hoven, Julia; Volkerling, Alexander; Bongers, Andre; Halliday, Glenda; Haass, Nikolas K; Kiernan, Matthew; Delerue, Fabien; Ittner, Lars M

    2015-11-01

    The nuclear transactive response DNA-binding protein 43 (TDP-43) undergoes relocalization to the cytoplasm with formation of cytoplasmic deposits in neurons in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Pathogenic mutations in the TDP-43-encoding TARDBP gene in familial ALS as well as non-mutant human TDP-43 have been utilized to model FTD/ALS in cell culture and animals, including mice. Here, we report novel A315T mutant TDP-43 transgenic mice, iTDP-43(A315T), with controlled neuronal over-expression. Constitutive expression of human TDP-43(A315T) resulted in pronounced early-onset and progressive neurodegeneration, which was associated with compromised motor performance, spatial memory and disinhibition. Muscle atrophy resulted in reduced grip strength. Cortical degeneration presented with pronounced astrocyte activation. Using differential protein extraction from iTDP-43(A315T) brains, we found cytoplasmic localization, fragmentation, phosphorylation and ubiquitination and insolubility of TDP-43. Surprisingly, suppression of human TDP-43(A315T) expression in mice with overt neurodegeneration for only 1 week was sufficient to significantly improve motor and behavioral deficits, and reduce astrogliosis. Our data suggest that functional deficits in iTDP-43(A315T) mice are at least in part a direct and transient effect of the presence of TDP-43(A315T). Furthermore, it illustrates the compensatory capacity of compromised neurons once transgenic TDP-43 is removed, with implications for future treatments.

  10. Effects of antagonists of growth hormone-releasing hormone (GHRH) on GH and insulin-like growth factor I levels in transgenic mice overexpressing the human GHRH gene, an animal model of acromegaly.

    Science.gov (United States)

    Kovacs, M; Kineman, R D; Schally, A V; Zarandi, M; Groot, K; Frohman, L A

    1997-11-01

    Transgenic mice overexpressing the human GH-releasing hormone (hGHRH) gene, an animal model of acromegaly, were used to investigate the effects of potent GHRH antagonists MZ-4-71 and MZ-5-156 on the excessive GH and insulin-like growth factor I (IGF-I) secretion caused by overproduction of hGHRH. Because metallothionein (MT)-GHRH mice express the hGHRH transgene in various tissues, including the pituitary and hypothalamus, initial experiments focused on the effectiveness of the GHRH antagonists in blocking basal and stimulated GH secretion from pituitary cells in vitro. Both MZ-4-71 and MZ-5-156 suppressed basal release of GH from superfused MT-GHRH pituitary cells, apparently by blocking the action of endogenously produced hGHRH. In addition, these antagonists effectively eliminated the response to stimulatory action of exogenous hGHRH(1-29)NH2 (30 and 100 nM). To ascertain whether MZ-4-71 and MZ-5-156 could antagonize the effect of hGHRH hyperstimulation in vivo, each antagonist was administered to MT-GHRH transgenic mice in a single iv dose of 10-200 microg. Both compounds decreased serum GH levels in transgenic mice by 39-72% at 1 h after injection. The inhibitory effect of 50 microg MZ-5-156 was maintained for 5 h. Twice daily ip administration of 100 microg MZ-5-156 for 3 days suppressed the highly elevated serum GH and IGF-I concentrations in transgenic mice by 56.8% and 39.0%, respectively. This treatment also reduced IGF-I messenger RNA levels in the liver by 21.8% but did not affect the level of GH messenger RNA in the pituitary. Our results demonstrate that GHRH antagonists MZ-4-71 and MZ-5-156 can inhibit elevated GH levels caused by overproduction of hGHRH. The suppression of circulating GH concentrations induced by the antagonists seems to be physiologically relevant, because both IGF-I secretion and synthesis also were reduced. Our findings, showing the suppression of GH and IGF-I secretion with GHRH antagonists, suggest that this class of analogs

  11. Ectopic expression of a cecropin transgene in the human malaria vector mosquito Anopheles gambiae (Diptera: Culicidae): effects on susceptibility to Plasmodium.

    Science.gov (United States)

    Kim, Won; Koo, Hyeyoung; Richman, Adam M; Seeley, Douglas; Vizioli, Jacopo; Klocko, Andrew D; O'Brochta, David A

    2004-05-01

    Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.

  12. Research advances on transgenic plant vaccines.

    Science.gov (United States)

    Han, Mei; Su, Tao; Zu, Yuan-Gang; An, Zhi-Gang

    2006-04-01

    In recent years, with the development of genetics molecular biology and plant biotechnology, the vaccination (e.g. genetic engineering subunit vaccine, living vector vaccine, nucleic acid vaccine) programs are taking on a prosperous evolvement. In particular, the technology of the use of transgenic plants to produce human or animal therapeutic vaccines receives increasing attention. Expressing vaccine candidates in vegetables and fruits open up a new avenue for producing oral/edible vaccines. Transgenic plant vaccine disquisitions exhibit a tempting latent exploiting foreground. There are a lot of advantages for transgenic plant vaccines, such as low cost, easiness of storage, and convenient immune-inoculation. Some productions converged in edible tissues, so they can be consumed directly without isolation and purification. Up to now, many transgenic plant vaccine productions have been investigated and developed. In this review, recent advances on plant-derived recombinant protein expression systems, infectious targets, and delivery systems are presented. Some issues of high concern such as biosafety and public health are also discussed. Special attention is given to the prospects and limitations on transgenic plant vaccines.

  13. High-level expressing YAC vector for transgenic animal bioreactors.

    Science.gov (United States)

    Fujiwara, Y; Miwa, M; Takahashi, R; Kodaira, K; Hirabayashi, M; Suzuki, T; Ueda, M

    1999-04-01

    The position effect is one major problem in the production of transgenic animals as mammary gland bioreactors. In the present study, we introduced the human growth hormone (hGH) gene into 210-kb human alpha-lactalbumin position-independent YAC vectors using homologous recombination and produced transgenic rats via microinjection of YAC DNA into rat embryos. The efficiency of producing transgenic rats with the YAC vector DNA was the same as that using plasmid constructs. All analyzed transgenic rats had one copy of the transgene and produced milk containing a high level of hGH (0.25-8.9 mg/ml). In transgenic rats with the YAC vector in which the human alpha-lactalbumin gene was replaced with the hGH gene, tissue specificity of hGH mRNA was the same as that of the endogenous rat alpha-lactalbumin gene. Thus, the 210-kb human alpha-lactalbumin YAC is a useful vector for high-level expression of foreign genes in the milk of transgenic animals.

  14. Generation of transgenic frogs.

    Science.gov (United States)

    Loeber, Jana; Pan, Fong Cheng; Pieler, Tomas

    2009-01-01

    The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed.

  15. Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle.

    Directory of Open Access Journals (Sweden)

    Ran Zhang

    Full Text Available 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.

  16. CD7 Positive Diffuse Large B-Cell Lymphoma Arising in a Background of Follicular Lymphoma: A Case Report and Review of the Literature

    Science.gov (United States)

    Rashidi, Hooman H.

    2016-01-01

    Diffuse large B-cell lymphoma (DLBCL) is a neoplasm of large B-lymphocytes with a diffuse growth pattern. The neoplastic cells express B-cell markers such as CD20 and PAX-5 and there may be coexpression of BCL-2, BCL-6, CD10, and MUM-1. With the exception of CD5, other T-cell markers are not commonly expressed in this neoplasm. Here, we describe the first reported case of a DLBCL with abnormal expression CD7 arising in a background of follicular lymphoma in an 81-year-old male who presented with a nontender left axillary mass. Additionally, no other T-cell antigens were expressed in this B-cell lymphoma. Expression of CD7 in DLBCL is exceptionally rare and its prognostic significance is unknown. Here, we describe this rare case with review of literature of known DLBCLs with expression of T-cell antigens.

  17. Towards Transgenic Primates: What can we learn from mouse genetics?

    Institute of Scientific and Technical Information of China (English)

    KUANG Hui; WANG Phillip L.; TSIEN Joe Z.

    2009-01-01

    Considering the great physiological and behavioral similarities with humans, monkeys represent the ideal models not only for the study of complex cognitive behavior but also for the precUnical research and development of novel therapeutics for treating human diseases. Various powerful genetic tech-nologies initially developed for making mouse models are being explored for generating transgenic primate models. We review the latest genetic engineering technologies and discuss the potentials and limitations for systematic production of transgenic primates.

  18. [Transgenic animals: uses and limitations in the 21st century medicine].

    Science.gov (United States)

    Cavagnari, Brian M

    2010-08-01

    One of the most important advances in biotechnology during the last decades was the development of transgenic animals. In this article, I discuss why transgenic animals are excellent models to analyze gen function and regulation, and to look for new therapeutic strategies for human diseases. Moreover, their use as bioreactors to produce pharmaceutical products for the treatment of human diseases, and the possibility of generating transgenic pigs as an alternative source of organ donors for humans is also discussed.

  19. Transgenic Crops for Herbicide Resistance

    Science.gov (United States)

    Since their introduction in 1995, crops made resistant to the broad-spectrum herbicides glyphosate and glufosinate with transgenes are widely available and used in much of the world. As of 2008, over 80% of the transgenic crops grown world-wide have this transgenic trait. This technology has had m...

  20. Transgene Stacking in Cotton Improvement

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To date,more and more transgenic varieties of upland cotton(Gossypium hirsutum L.) generated with transgenes,which derived from varies of alien species,are playing important role in agricultural production.Stacking of multi-transgenes has a potential for combining all the merits of distinct

  1. Transgenic Farm Animals

    Science.gov (United States)

    The development of recombinant DNA technology has enabled scientists to isolate single genes, analyze and modify their nucleotide structure(s), make copies of these isolated genes, and insert copies of these genes into the genome of plants and animals. The transgenic technology of adding genes to li...

  2. [Progress on transgenic mosquitoes].

    Science.gov (United States)

    Yang, Pin

    2011-04-30

    The genetically modified mosquitoes have been developed aiming to control mosquito-borne diseases by either reducing population sizes or replacing existing populations with vectors unable to transmit the disease. introduces some progress on the generation of transgenic mosquitoes and their fitness in wild population. This paper

  3. Spatial and temporal control of transgene expression in zebrafish.

    Directory of Open Access Journals (Sweden)

    Alexander A Akerberg

    Full Text Available Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAS-controlled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments.

  4. Reduction of choline acetyltransferase activities in APP770 transgenic mice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Transgenic mice overexpressing the 770-amino acid isoform of human Alzheimer amyloid precursor protein exhibit extracellular b -amyloid deposits in brain regions including cerebral cortex and hippocampus, which are severely affected in Alzheimer's disease patients. Significant reduction in choline acetyltransferase (ChAT) activities has been observed in both cortical and hippocampal brain regions in the transgenic mice at the age of 10 months compared with the age-matched non-transgenic mice, but such changes have not been observed in any brain regions of the transgenic mice under the age of 5 months. These results suggest that deposition of b -amyloid can induce changes in the brain cholinergic system of the transgenic mice.

  5. Transgenic dairy cattle: genetic engineering on a large scale.

    Science.gov (United States)

    Wall, R J; Kerr, D E; Bondioli, K R

    1997-09-01

    Amid the explosion of fundamental knowledge generated from transgenic animal models, a small group of scientists has been producing transgenic livestock with goals of improving animal production efficiency and generating new products. The ability to modify mammary-specific genes provides an opportunity to pursue several distinctly different avenues of research. The objective of the emerging gene "pharming" industry is to produce pharmaceuticals for treating human diseases. It is argued that mammary glands are an ideal site for producing complex bioactive proteins that can be cost effectively harvested and purified. Consequently, during the past decade, approximately a dozen companies have been created to capture the US market for pharmaceuticals produced from transgenic bioreactors estimated at $3 billion annually. Several products produced in this way are now in human clinical trials. Another research direction, which has been widely discussed but has received less attention in the laboratory, is genetic engineering of the bovine mammary gland to alter the composition of milk destined for human consumption. Proposals include increasing or altering endogenous proteins, decreasing fat, and altering milk composition to resemble that of human milk. Initial studies using transgenic mice to investigate the feasibility of enhancing manufacturing properties of milk have been encouraging. The potential profitability of gene "pharming" seems clear, as do the benefits of transgenic cows producing milk that has been optimized for food products. To take full advantage of enhanced milk, it may be desirable to restructure the method by which dairy producers are compensated. However, the cost of producing functional transgenic cattle will remain a severe limitation to realizing the potential of transgenic cattle until inefficiencies of transgenic technology are overcome. These inefficiencies include low rates of gene integration, poor embryo survival, and unpredictable transgene

  6. Expression systems and species used for transgenic animal bioreactors.

    Science.gov (United States)

    Wang, Yanli; Zhao, Sihai; Bai, Liang; Fan, Jianglin; Liu, Enqi

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon), the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow) that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

  7. Expression Systems and Species Used for Transgenic Animal Bioreactors

    Directory of Open Access Journals (Sweden)

    Yanli Wang

    2013-01-01

    Full Text Available Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm cocoon, the mammary glands of transgenic animals have enormous potential. Compared with other mammalian species (pig, goat, sheep, and cow that are currently being studied as bioreactors, rabbits offer many advantages: high fertility, easy generation of transgenic founders and offspring, insensitivity to prion diseases, relatively high milk production, and no transmission of severe diseases to humans. Noticeably, for a small- or medium-sized facility, the rabbit system is ideal to produce up to 50 kg of protein per year, considering both economical and hygienic aspects; rabbits are attractive candidates for the mammary-gland-specific expression of recombinant proteins. We also reviewed recombinant proteins that have been produced by targeted expression in the mammary glands of rabbits and discussed the limitations of transgenic animal bioreactors.

  8. Effect of transgenic human lactoferrin goats' milk on growth and metabolic performance in rats%转人乳铁蛋白基因山羊乳对大鼠生长及代谢机能的影响

    Institute of Scientific and Technical Information of China (English)

    王二先; 徐旭俊; 苗晋锋; 陈建泉; 成国祥; 杨倩

    2011-01-01

    选用30只100 g左右清洁级SD大鼠,雌雄各半,随机均分为3组,转基因乳组每天2次灌胃转人乳铁蛋白基因(hLF)波尔山羊乳1 mL(蛋白含量0.93 g·mL-1),普通乳组灌胃未经任何处理的普通波尔山羊乳1 mL(蛋白浓度0.93 g·mL-1),生理盐水组灌胃等量灭菌生理盐水;自由采食、饮水,连续15 d;颈部静脉采血致死,收集血清.结果显示:灌胃转hLF基因山羊乳显著提高了雌、雄大鼠的日增重,与生理盐水组相比差异极显著(P0.05).试验结果表明:短期灌胃转hLF基因山羊乳能够促进大鼠的生长,但对大鼠的代谢机能没有显著影响.%Fifteen healthy SD female rats and fifteen healthy male rats weighting about 100 g were randomly distributed irnto three groups. The transgenic milk group was infused with transgenic human lactoferrin gene( hLF) goats' milk 1 mL( protein concentration at 0.93 g·mL-1 ) ,directly into the gastric two times a day ;the normal milk group with normal goats' milk( protein concentration at O. 93 g·mL-1 ) ,and the physiological saline group with sterile pyrogen-free physiological saline at the same quantity. Three groups were provided a standard rodent chow and an ad libitum supply of water. After 15 days of infusing, all the rats were killed by collecting living body blood in venous of neck. The result indicated that the average daily gain of the female and male rats which were infused with transgenic hLF goats' milk increased in evidence compared to the physiological saline group(P<0.01 )and the normal milk group( P<O. 05 ). However,there were no difference between the normal milk group and the physiological saline group. And the other measurements and biochemical examination of the transgenic milk group and the normal milk group didn't change significantly compared to the physiological saline group( P>O. 05 ), moreover, no obvious change were observed between the transgenic and normal milk group. Overall, the result of the present

  9. Cloning of Human Germinal Center Kinase Gene and Construction of Its Transgenic Cells%人生发中心激酶基因克隆及其转基因细胞的构建

    Institute of Scientific and Technical Information of China (English)

    谈勤春; 周璇; 张学光

    2011-01-01

    Objective To clone human germinal center kinase (GCK) gene and construct recombinant plasmid vector carrying the target gene which can be expressed stably in mammal cell line HEK293.Methods Human GCK gene was amplified by PCR using the pCMV5-GCK as template and then confirmed by DNA-sequence analysis. Digested with the restriction endonucleases Bgl Ⅱ and SalI,the human GCK gene was inserted into pIRES2-EGFP plasmid vector. The recombinant pIRES2-EGFP plasmid vector was cotransfected into the HEK293 cell in the context of Lipfect2000. After 72 hours, HEK293 cell line stably expressing human DC-SIGN protein was selected in the presence of G418. Result The fulllength of human GCK gene was cloned and the recombinant pIRES2-EGFP plasmid vector was constructed, the HEK293 cell line expressing human GCK was successfully transfected and selected. Results of RT-PCR and Western blot indicated that HEK293 transgenic cells could stably express human GCK protein. Conclusion Cloning of human GCK gene and construction of the recombinant plRES2-EGFP plasmid vector and HEK293 transgenic cell line stably expressing GCK protein could contribute to further biological function research.%目的 克隆人生发中心激酶(GCK)基因,并构建含有该目的基因的plRES2-EGFP重组质粒载体,获得稳定表达GCK分子的基因转染细胞.方法 采用RT-PCR法从pCMV5-GCK质粒载体上克隆GCK基因,通过双酶切(BglⅡ,Sall)装入plRES2-EGFP质粒载体中,脂质体法共转染HEK293细胞,24 h后加入G418进行筛选,挑选出能稳定表达GCK蛋白的HEK293细胞株.结果 构建了用于表达的含GCK基因的plRES2-EGFP质粒载体,继而经RT-PCR、Western blot检测筛选出稳定表达人GCK蛋白的HEK293转基因细胞.结论 构建了含人GCK基因重组plRES2-EGFP质粒载体和稳定表达人GCK蛋白的细胞株,为该基因功能的后续研究奠定了基础.

  10. Effects of Human Alpha-Synuclein A53T-A30P Mutations on SVZ and Local Olfactory Bulb Cell Proliferation in a Transgenic Rat Model of Parkinson Disease

    Directory of Open Access Journals (Sweden)

    Faustine Lelan

    2011-01-01

    Full Text Available A transgenic Sprague Dawley rat bearing the A30P and A53T α-synuclein (α-syn human mutations under the control of the tyrosine hydroxylase promoter was generated in order to get a better understanding of the role of the human α-syn mutations on the neuropathological events involved in the progression of the Parkinson’s disease (PD. This rat displayed olfactory deficits in the absence of motor impairments as observed in most early PD cases. In order to investigate the role of the mutated α-syn on cell proliferation, we focused on the subventricular zone (SVZ and the olfactory bulbs (OB as a change of the proliferation could affect OB function. The effect on OB dopaminergic innervation was investigated. The human α-syn co-localized in TH-positive OB neurons. No human α-syn was visualized in the SVZ. A significant increase in resident cell proliferation in the glomerular but not in the granular layers of the OB and in the SVZ was observed. TH innervation was significantly increased within the glomerular layer without an increase in the size of the glomeruli. Our rat could be a good model to investigate the role of human mutated α-syn on the development of olfactory deficits.

  11. Selenoprotein-Transgenic Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Jiazuan Ni

    2013-02-01

    Full Text Available Selenium (Se deficiency is associated with the occurrence of many diseases. However, excessive Se supplementation, especially with inorganic Se, can result in toxicity. Selenoproteins are the major forms of Se in vivo to exert its biological function. Expression of those selenoproteins, especially with the application of a newly developed system, is thus very important for studying the mechanism of Se in nutrition. The use of Chlamydomonas reinhardtii (C. reinhardtii as a biological vector to express an heterogeneous protein is still at the initial stages of development. In order to investigate the possibility of using this system to express selenoproteins, human 15-KDa selenoprotein (Sep15, a small but widely distributed selenoprotein in mammals, was chosen for the expression platform test. Apart from the wild-type human Sep15 gene fragment, two Sep15 recombinants were constructed containing Sep15 open reading frame (ORF and the selenocysteine insertion sequence (SECIS element from either human Sep15 or C. reinhardtii selenoprotein W1, a highly expressed selenoprotein in this alga. Those Sep15-containing plasmids were transformed into C. reinhardtii CC-849 cells. Results showed that Sep15 fragments were successfully inserted into the nuclear genome and expressed Sep15 protein in the cells. The transgenic and wild-type algae demonstrated similar growth curves in low Se culture medium. To our knowledge, this is the first report on expressing human selenoprotein in green alga.

  12. Simple and Efficient Method for Measuring Anti-Toxoplasma Immunoglobulin Antibodies in Human Sera Using Complement-Mediated Lysis of Transgenic Tachyzoites Expressing β-Galactosidase

    Science.gov (United States)

    Dando, Caroline; Gabriel, Katie E.; Remington, Jack S.; Parmley, Stephen F.

    2001-01-01

    A simple and efficient method using transgenic Toxoplasma gondii tachyzoites expressing β-galactosidase was developed for detection of specific antibodies against the parasite in sera of patients. The titers obtained with the new test were similar to those obtained with the Sabin-Feldman dye test run in parallel. Although significant changes in endpoint titers were not observed when sera drawn sequentially at 2- to 3-week intervals were tested with both procedures, apparent differences in antibody affinity were observed with the new test which were not perceptible with the Sabin-Feldman dye test. Like the Sabin-Feldman dye test, the new test is based on complement lysis of tachyzoites, but it is much easier to perform and the reaction is read colorimetrically instead of visually. PMID:11376045

  13. Transgenic animal bioreactors in biotechnology and production of blood proteins.

    Science.gov (United States)

    Lubon, H

    1998-01-01

    The regulatory elements of genes used to target the tissue-specific expression of heterologous human proteins have been studied in vitro and in transgenic mice. Hybrid genes exhibiting the desired performance have been introduced into large animals. Complex proteins like protein C, factor IX, factor VIII, fibrinogen and hemoglobin, in addition to simpler proteins like alpha 1-antitrypsin, antithrombin III, albumin and tissue plasminogen activator have been produced in transgenic livestock. The amount of functional protein secreted when the transgene is expressed at high levels may be limited by the required posttranslational modifications in host tissues. This can be overcome by engineering the transgenic bioreactor to express the appropriate modifying enzymes. Genetically engineered livestock are thus rapidly becoming a choice for the production of recombinant human blood proteins.

  14. Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo.

    Science.gov (United States)

    Jiang, Weiwu; Couroucli, Xanthi I; Wang, Lihua; Barrios, Roberto; Moorthy, Bhagavatula

    2011-04-01

    Supplemental oxygen administration is frequently administered to pre-term and term infants having pulmonary insufficiency. However, hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Cytochrome P450 (CYP)A enzymes have been implicated in hyperoxic lung injury. In this study, we tested the hypothesis that hyperoxia induces CYP1A1 and 1A2 enzymes by transcriptional activation of the corresponding promoters in vivo, and transgenic mice expressing the human CYP1A1 or the mouse 1A2 promoter would be more susceptible to hyperoxic lung injury than wild type (WT) mice. Adult WT (CD-1) (12week-old) mice, transgenic mice carrying a 10kb human CYP1A1 promoter and the luciferase (luc) reporter gene (CYP1A1-luc), or mice expressing the mouse CYP1A2 promoter (CYP1A2-luc) were maintained in room air or exposed to hyperoxia for 24-72h. Hyperoxia exposure of CYP1A1-luc mice for 24 and 48h resulted in 2.5- and 1.25-fold increases, respectively, in signal intensities, compared to room air controls. By 72h, the induction had declined to control levels. CYP1A2-luc mice also showed enhanced luc expression after 24-48h, albeit to a lesser extent than those expressing the CYP1A1 promoter. Also, these mice showed decreased levels of endogenous CYP1A1 and 1A2 expression after prolonged hyperoxia, and were also more susceptible to lung injury than similarly exposed WT mice, with CYP1A2-luc mice showing the greatest injury. Our results support the hypothesis that hyperoxia induces CYP1A enzymes by transcriptional activation of its corresponding promoters, and that decreased endogenous expression of these enzymes contribute to the increased susceptibilities to hyperoxic lung injury in the transgenic animals. In summary, this is the first report providing direct evidence of hyperoxia-mediated induction of CYP1A1 and CYP1A2 expression in vivo by mechanisms entailing transcriptional activation of the corresponding promoters, a phenomenon that has

  15. Expression of the human UGT1 locus in transgenic mice by 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY-14643) and implications on drug metabolism through peroxisome proliferator-activated receptor alpha activation.

    Science.gov (United States)

    Senekeo-Effenberger, Kathy; Chen, Shujuan; Brace-Sinnokrak, Erin; Bonzo, Jessica A; Yueh, Mei-Fei; Argikar, Upendra; Kaeding, Jenny; Trottier, Jocelyn; Remmel, Rory P; Ritter, Joseph K; Barbier, Olivier; Tukey, Robert H

    2007-03-01

    The UDP-glucuronosyltransferase (UGT) 1A genes in humans have been shown to be differentially regulated in a tissue-specific fashion. Transgenic mice carrying the human UGT1 locus (Tg-UGT1) were recently created, demonstrating that expression of the nine UGT1A genes closely resembles the patterns of expression observed in human tissues. In the present study, UGT1A1, UGT1A3, UGT1A4, and UGT1A6 have been identified as targets of the peroxisome proliferator-activated receptor (PPAR) alpha in human hepatocytes and Tg-UGT1 mice. Oral administration of the PPARalpha agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (pirinixic acid, WY-14643) to Tg-UGT1 mice led to induction of these proteins in either the liver, gastrointestinal tract, or kidney. The levels of induced UGT1A3 gene transcripts in liver and UGT1A4 protein in small intestine correlated with induced lamotrigine glucuronidation activity in these tissues. With UGT1A3 previously identified as the major human enzyme involved in human C24-glucuronidation of lithocholic acid (LCA), the dramatic induction of liver UGT1A3 RNA in Tg-UGT1 mice was consistent with the formation of LCA-24G in plasma. Furthermore, PPAR-responsive elements (PPREs) were identified flanking the UGT1A1, UGT1A3, and UGT1A6 genes by a combination of site-directed mutagenesis, specific binding to PPARalpha and retinoic acid X receptor alpha, and functional response of the concatenated PPREs in HepG2 cells overexpressing PPARalpha. In conclusion, these results suggest that oral fibrate treatment in humans will induce the UGT1A family of proteins in the gastrointestinal tract and liver, influencing bile acid glucuronidation and first-pass metabolism of other drugs that are taken concurrently with hypolipidemic therapy.

  16. Transgenic algae engineered for higher performance

    Energy Technology Data Exchange (ETDEWEB)

    Unkefer, Pat J; Anderson, Penelope S; Knight, Thomas J

    2014-10-21

    The present disclosure relates to transgenic algae having increased growth characteristics, and methods of increasing growth characteristics of algae. In particular, the disclosure relates to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and to transgenic algae comprising a glutamine phenylpyruvate transaminase transgene and a glutamine synthetase.

  17. [Application of ICP-MS to the detection of heavy metals in transgenic corn].

    Science.gov (United States)

    Rui, Yu-Kui; Guo, Jing; Huang, Kun-Lun; Jin, Yin-Hua; Luo, Yun-Bo

    2007-04-01

    With the rapid development of the transgenic food, more and more transgenic food has been pouring into the market attracting much attention to the transgenic food's edible safety. Transgenic corns and its parents were studied by ICP-MS to detect the heavy metals. The results showed that the transgenic corn accumulated less heavy metals (Ni, Cu, Cd, As, Cr, Zn and Hg) than their own parents; and the contents of some heavy metals (V, Co and Pb) in transgenic corns were similar to their parents. All the data showed that the insertion of foreign gene (Bt) might change the absorbing dynamics of most heavy metals, especially some important heavy metals, which are disadvantageous to human health. The present paper indicated that the change in heavy metals absorption could harm the edible safety of transgenic plant. The cause of this change should be studied further.

  18. The effect of mammary gland-specific transgene expression on rabbit reproductive gland structure.

    Science.gov (United States)

    Tvrdá, Eva; Massányi, Peter; Lukác, Norbert; Danko, Ján; Schlarmannova, Janka; Chrenek, Peter

    2014-01-01

    Transgenic rabbits are excellent animal models for human diseases and suitable bioreactors for the production of recombinant proteins on an experimental and commercial scale. The aim of this study was to compare the structure of the mWAP-hFVIII transgenic and non-transgenic rabbit ovarian and testicular tissue. Ovarian and testicular tissue samples were taken from transgenic and non-transgenic New Zealand White rabbits, examined by optical microscopy and analyzed morphometrically. An increase of the relative volume of primary follicles and a decrease of the relative volume of antral follicles was detected in the transgenic ovarian structure (P 0.05). In the testes a significant decrease (P transgenic testicular structure, but the relative volume of all basic structures (germinal epithelium, interstitium and lumen) was unaltered (P > 0.05). Generally, this study demonstrates a weak negative effect of mWAP-hFVIII transgenesis on rabbit gonadal structure.

  19. Functional imaging of the angiogenic switch in a transgenic mouse model of human breast cancer by dynamic contrast enhanced magnetic resonance imaging.

    Science.gov (United States)

    Consolino, Lorena; Longo, Dario Livio; Dastrù, Walter; Cutrin, Juan Carlos; Dettori, Daniela; Lanzardo, Stefania; Oliviero, Salvatore; Cavallo, Federica; Aime, Silvio

    2016-07-15

    Tumour progression depends on several sequential events that include the microenvironment remodelling processes and the switch to the angiogenic phenotype, leading to new blood vessels recruitment. Non-invasive imaging techniques allow the monitoring of functional alterations in tumour vascularity and cellularity. The aim of this work was to detect functional changes in vascularisation and cellularity through Dynamic Contrast Enhanced (DCE) and Diffusion Weighted (DW) Magnetic Resonance Imaging (MRI) modalities during breast cancer initiation and progression of a transgenic mouse model (BALB-neuT mice). Histological examination showed that BALB-neuT mammary glands undergo a slow neoplastic progression from simple hyperplasia to invasive carcinoma, still preserving normal parts of mammary glands. DCE-MRI results highlighted marked functional changes in terms of vessel permeability (K(trans) , volume transfer constant) and vascularisation (vp , vascular volume fraction) in BALB-neuT hyperplastic mammary glands if compared to BALB/c ones. When breast tissue progressed from simple to atypical hyperplasia, a strong increase in DCE-MRI biomarkers was observed in BALB-neuT in comparison to BALB/c mice (K(trans)  = 5.3 ± 0.7E-4 and 3.1 ± 0.5E-4; vp  = 7.4 ± 0.8E-2 and 4.7 ± 0.6E-2 for BALB-neuT and BALB/c, respectively) that remained constant during the successive steps of the neoplastic transformation. Consistent with DCE-MRI observations, microvessel counting revealed a significant increase in tumour vessels. Our study showed that DCE-MRI estimates can accurately detect the angiogenic switch at early step of breast cancer carcinogenesis. These results support the view that this imaging approach is an excellent tool to characterize microvasculature changes, despite only small portions of the mammary glands developed neoplastic lesions in a transgenic mouse model.

  20. Transgenic nonhuman primates for neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    Chan Anthony WS

    2004-06-01

    Full Text Available Abstract Animal models that represent human diseases constitute an important tool in understanding the pathogenesis of the diseases, and in developing effective therapies. Neurodegenerative diseases are complex disorders involving neuropathologic and psychiatric alterations. Although transgenic and knock-in mouse models of Alzheimer's disease, (AD, Parkinson's disease (PD and Huntington's disease (HD have been created, limited representation in clinical aspects has been recognized and the rodent models lack true neurodegeneration. Chemical induction of HD and PD in nonhuman primates (NHP has been reported, however, the role of intrinsic genetic factors in the development of the diseases is indeterminable. Nonhuman primates closely parallel humans with regard to genetic, neuroanatomic, and cognitive/behavioral characteristics. Accordingly, the development of NHP models for neurodegenerative diseases holds greater promise for success in the discovery of diagnoses, treatments, and cures than approaches using other animal species. Therefore, a transgenic NHP carrying a mutant gene similar to that of patients will help to clarify our understanding of disease onset and progression. Additionally, monitoring disease onset and development in the transgenic NHP by high resolution brain imaging technology such as MRI, and behavioral and cognitive testing can all be carried out simultaneously in the NHP but not in other animal models. Moreover, because of the similarity in motor repertoire between NHPs and humans, it will also be possible to compare the neurologic syndrome observed in the NHP model to that in patients. Understanding the correlation between genetic defects and physiologic changes (e.g. oxidative damage will lead to a better understanding of disease progression and the development of patient treatments, medications and preventive approaches for high risk individuals. The impact of the transgenic NHP model in understanding the role which

  1. Ethical issues in transgenics.

    Science.gov (United States)

    Sherlock, R; Morrey, J D

    2000-01-01

    The arguments of critics and concerns of the public on generating transgenic cloned animals are analyzed for the absence or presence of logical structure. Critics' arguments are symbolically compared with "genetic trespassing," "genetic speeding," or "going the wrong way," and responses are provided to these arguments. Scientists will be empowered to participate in the public discussion and to engage the critics on these issues as they consider thoughtful, plausible responses to their concerns. Temporary moratoriums are recognized as a plausible approach to dealing with possible concerns of new scientific advancements.

  2. Detection of the Integration of Human Transferrin Gene on Chromosomes in Transgenic Cloned Cows%人转铁蛋白转基因克隆牛整合位点检测

    Institute of Scientific and Technical Information of China (English)

    刘宇; 朱怡文; 许淼; 黄英

    2012-01-01

    为检测外源基因整合在转基因克隆牛染色体上的定位情况,通过优化染色体荧光原位杂交技术,成功对4头人转铁蛋白转基因克隆牛的整合位点进行定位.每头牛分析中期分裂相40个以上,杂交率在53.1%~86.8%之间,且杂交信号分别位于转基因克隆牛的11号、15号和19号染色体的相应位置;另外采用定量PCR方法对杂交信号强弱不同的转基因克隆牛个体进行整合位点拷贝数的检测,结果显示拷贝数的多少与杂交信号强弱直接有关.%To detect the integration of human transferrin gene(hTF) on chromosomes in transgenic cloned cows.the author optimized fluorescence in situ hybridization(FISH). For transgenic cloned cows,53. 1% to 86. 8% of metaphases showed hybridization signals which were located on different chromosomes,such as Chr. 11, Chr. 15 and Chr. 19. The results showed that using FISH to detect the integration site and hybridization signals was highly efficient and specific,also the copy numbers were detected by Real-time PCR.

  3. DRPLA transgenic mouse substrains carrying single copy of full-length mutant human DRPLA gene with variable sizes of expanded CAG repeats exhibit CAG repeat length- and age-dependent changes in behavioral abnormalities and gene expression profiles.

    Science.gov (United States)

    Suzuki, Kazushi; Zhou, Jiayi; Sato, Toshiya; Takao, Keizo; Miyagawa, Tsuyoshi; Oyake, Mutsuo; Yamada, Mitunori; Takahashi, Hitoshi; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

    2012-05-01

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant progressive neurodegenerative disorder with intellectual deterioration and various motor deficits including ataxia, choreoathetosis, and myoclonus, caused by an abnormal expansion of CAG repeats in the DRPLA gene. Longer expanded CAG repeats contribute to an earlier age of onset, faster progression, and more severe neurological symptoms in DRPLA patients. In this study, we have established DRPLA transgenic mouse lines (sublines) harboring a single copy of the full-length mutant human DRPLA gene carrying various lengths of expanded CAG repeats (Q76, Q96, Q113, and Q129), which have clearly shown motor deficits and memory disturbance whose severity increases with the length of expanded CAG repeats and age, and successfully replicated the CAG repeat length- and age-dependent features of DRPLA patients. Neuronal intranuclear accumulation of the mutant DRPLA protein has been suggested to cause transcriptional dysregulation, leading to alteration in gene expression and neuronal dysfunction. In this study, we have conducted a comprehensive analysis of gene expression profiles in the cerebrum and cerebellum of transgenic mouse lines at 4, 8, and 12 weeks using multiple microarray platforms, and demonstrated that both the number and expression levels of the altered genes are highly dependent on CAG repeat length and age in both brain regions. Specific groups of genes and their function categories were identified by further agglomerative cluster analysis and gene functional annotation analysis. Calcium signaling and neuropeptide signaling, among others, were implicated in the pathophysiology of DRPLA. Our study provides unprecedented CAG-repeat-length-dependent mouse models of DRPLA, which are highly valuable not only for elucidating the CAG-repeat-length-dependent pathophysiology of DRPLA but also for developing therapeutic strategies for DRPLA.

  4. Containment and competition: transgenic animals in the One Health agenda.

    Science.gov (United States)

    Lezaun, Javier; Porter, Natalie

    2015-03-01

    The development of the One World, One Health agenda coincides in time with the appearance of a different model for the management of human-animal relations: the genetic manipulation of animal species in order to curtail their ability as carriers of human pathogens. In this paper we examine two examples of this emergent transgenic approach to disease control: the development of transgenic chickens incapable of shedding avian flu viruses, and the creation of transgenic mosquitoes refractory to dengue or malaria infection. Our analysis elaborates three distinctions between the One World, One Health agenda and its transgenic counterpoint. The first concerns the conceptualization of outbreaks and the forms of surveillance that support disease control efforts. The second addresses the nature of the interspecies interface, and the relative role of humans and animals in preventing pathogen transmission. The third axis of comparison considers the proprietary dimensions of transgenic animals and their implications for the assumed public health ethos of One Health programs. We argue that the fundamental difference between these two approaches to infectious disease control can be summarized as one between strategies of containment and strategies of competition. While One World, One Health programs seek to establish an equilibrium in the human-animal interface in order to contain the circulation of pathogens across species, transgenic strategies deliberately trigger a new ecological dynamic by introducing novel animal varieties designed to out-compete pathogen-carrying hosts and vectors. In other words, while One World, One Health policies focus on introducing measures of inter-species containment, transgenic approaches derive their prophylactic benefit from provoking new cycles of intra-species competition between GM animals and their wild-type counterparts. The coexistence of these divergent health protection strategies, we suggest, helps to elucidate enduring tensions and

  5. Efficient production of transgenic melon via Agrobacterium-mediated transformation.

    Science.gov (United States)

    Bezirganoglu, I; Hwang, S Y; Shaw, J F; Fang, T J

    2014-04-25

    Oriental melon (Cucumis melo L. var. makuwa) is an important fruit for human consumption. However, this plant species is one of the most recalcitrant to genetic transformation. The lack of an efficient in vitro system limits the development of a reproducible genetic transformation protocol for Oriental melon. In this study, an efficient transgenic production method for Agrobacterium-mediated transformation using cotyledon explants of Oriental melon was developed. Cotyledon explants were pre-cultivated for two days in the dark, and the optimal conditions for transformation of melon were determined to be a bacteria concentration of OD600 0.6, inoculation for 30 min, and two days of co-cultivation. Transgenic melon plants were produced from kanamycin-resistant shoots. A total of 11 independent transgenic plants were regenerated with a transformation efficiency of 0.8% of the inoculated explants. The transgenic plants were phenotypically normal and fully fertile, which might be a consequence of the co-cultivation time.

  6. Epigenetic silencing in transgenic plants

    Directory of Open Access Journals (Sweden)

    Sarma eRajeev Kumar

    2015-09-01

    Full Text Available Epigenetic silencing is a natural phenomenon in which the expression of gene is regulated through modifications of DNA, RNA or histone proteins. It is a mechanism for defending host genomes against the effects of transposable element, viral infection and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was discovery of silencing in transgenic tobacco plants due to interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, i.e. transcriptional gene silencing (TGS, which is associated with heavy methylation of promoter regions and blocks the transcription of transgene. The basic mechanism underlying post-transcriptional gene silencing (PTGS is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of a major concern in transgenic technology used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes are required. The current status of epigenetic silencing in transgenic technology has been discussed and summarized in this mini-review.

  7. Epigenetic silencing in transgenic plants

    Science.gov (United States)

    Rajeevkumar, Sarma; Anunanthini, Pushpanathan; Sathishkumar, Ramalingam

    2015-01-01

    Epigenetic silencing is a natural phenomenon in which the expression of genes is regulated through modifications of DNA, RNA, or histone proteins. It is a mechanism for defending host genomes against the effects of transposable elements and viral infection, and acts as a modulator of expression of duplicated gene family members and as a silencer of transgenes. A major breakthrough in understanding the mechanism of epigenetic silencing was the discovery of silencing in transgenic tobacco plants due to the interaction between two homologous promoters. The molecular mechanism of epigenetic mechanism is highly complicated and it is not completely understood yet. Two different molecular routes have been proposed for this, that is, transcriptional gene silencing, which is associated with heavy methylation of promoter regions and blocks the transcription of transgenes, and post-transcriptional gene silencing (PTGS), the basic mechanism is degradation of the cytosolic mRNA of transgenes or endogenous genes. Undesired transgene silencing is of major concern in the transgenic technologies used in crop improvement. A complete understanding of this phenomenon will be very useful for transgenic applications, where silencing of specific genes is required. The current status of epigenetic silencing in transgenic technology is discussed and summarized in this mini-review. PMID:26442010

  8. Intestinal carcinogenesis of two food processing contaminants, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 5-hydroxymethylfurfural, in transgenic FVB min mice expressing human sulfotransferases.

    Science.gov (United States)

    Svendsen, Camilla; Meinl, Walter; Glatt, Hansruedi; Alexander, Jan; Knutsen, Helle K; Hjertholm, Hege; Rasmussen, Tone; Husøy, Trine

    2012-12-01

    Humans express sulfotransferases (SULTs) of the SULT1A subfamily in many tissues, whilst the single SULT1A gene present in rodents is mainly expressed in liver. The food processing contaminants, 5-hydroxymethylfurfural (HMF) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are bioactivated by human SULT1A1 and SULT1A2. FVB multiple intestinal neoplasia (Min) mice, which spontaneously develop tumors and flat aberrant crypt foci (ACF) in intestine, were crossed with transgenic FVB mice expressing human SULT1A1 and 1A2 (hSULT) in several tissues, giving rise to wild-type and Min mice with and without hSULT. One-week-old Min mice with or without hSULT were given HMF (375 or 750 mg/kg bw) or saline by gavage three times a week for 11 wk. In another experiment, the F1 generation received subcutaneous injections of 50 mg/kg bw PhIP or saline 1 wk before birth, and 1, 2, and 3 wk after birth. HMF did not affect the formation of tumors, but may have induced some flat ACF (incidence 15-20%) in Min mice with and without hSULT. No control mouse developed any flat ACF. With the limitation that these putative effects were weak, they were unaffected by hSULT expression. The carcinogenic effect of PhIP increased in the presence of hSULT, with a significant increase in both incidence (31-80%) and number of colonic tumors (0.4-1.3 per animal). Thus, intestinal expression of human SULT1A1 and 1A2 might increase the susceptibility to compounds bioactivated via this pathway implying that humans might be more susceptible than conventional rodent models.

  9. Green fluorescent protein (GFP) transgenic pig produced by somatic cell nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    LIU ZhongHua; SUN Shuang; LI YuTian; WANG HongBin; R S PRATHER; SONG Jun; WANG ZhenKun; TIAN JiangTian; KONG QingRan; ZHENG Zhong; YIN Zhi; GAO Li; MA HaiKun

    2008-01-01

    Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm ani-mals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfection system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe-tal-derived fibroblast cells cultured with 4.0 plJmL liposome and 1.6 pg/mL plasmid DNA for 6 h re-sulted in the highest transfection rate (3.6%). The percentage of GFP reconstructed embryos that de-veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Re-constructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 de-livered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.

  10. Pituitary mammosomatotroph adenomas develop in old mice transgenic for growth hormone-releasing hormone

    DEFF Research Database (Denmark)

    Asa, S L; Kovacs, K; Stefaneanu, L

    1990-01-01

    It has been shown that mice transgenic for human growth hormone-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs and mammosomatotrophs, cells capable of producing both growth hormone and prolactin, by 8 months of age. We now report for the first time that old GRH-transgenic m......-transgenic mice, 16 to 24 months of age, develop pituitary mammosomatotroph adenomas. These findings provide conclusive evidence that protracted stimulation of secretory activity can cause proliferation, hyperplasia and adenoma of adenohypophysial cells....

  11. Efficient and Rapid Development of Transgenic Hamster Models of TSEs Using a Radical New Technology

    Science.gov (United States)

    2007-09-01

    by TOSK Inc. to introduce the human, sheep, and other prion genes into golden Syrian hamsters and to use the new transgenic animals both to measure...Transgenic Hamster Models of TSEs Using a Radical New Technology PRINCIPAL INVESTIGATOR: Robert G. Rohwer, Ph.D. Irena Alexeeva, Ph.D...From - To) 1 SEP 2003 - 31 AUG 2007 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Efficient and Rapid Development of Transgenic Hamster Models of

  12. Transgenic Studies with a Keratin Promoter-Driven Growth Hormone Transgene: Prospects for Gene Therapy

    Science.gov (United States)

    Wang, Xiaoming; Zinkel, Sandra; Polonsky, Kenneth; Fuchs, Elaine

    1997-01-01

    Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈ 1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.

  13. Luteinizing Hormone Receptor Knockout (LuRKO) Mice and Transgenic Human Chorionic Gonadotropin (hCG)-Overexpressing Mice (hCG αβ+) Have Bone Phenotypes

    National Research Council Canada - National Science Library

    Yarram, S. J; Perry, M. J; Christopher, T. J; Westby, K; Brown, N. L; Lamminen, T; Rulli, S. B; Zhang, F.-P; Huhtaniemi, I; Sandy, J. R; Mansell, J. P

    2003-01-01

    Considerable attention has been paid to the role of sex steroids during periods of major skeletal turnover, but the interaction of the gonadotropic hormones, which include LH, FSH, and human chorionic gonadotropin (hCG...

  14. Increased susceptibility of transgenic mice expressing human PrP to experimental sheep bovine spongiform encephalopathy is not due to increased agent titre in sheep brain tissue.

    Science.gov (United States)

    Plinston, Chris; Hart, Patricia; Hunter, Nora; Manson, Jean C; Barron, Rona M

    2014-08-01

    Bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt-Jakob disease in humans have previously been shown to be caused by the same strain of transmissible spongiform encephalopathy agent. It is hypothesized that the agent spread to humans following consumption of food products prepared from infected cattle. Despite evidence supporting zoonotic transmission, mouse models expressing human prion protein (HuTg) have consistently shown poor transmission rates when inoculated with cattle BSE. Higher rates of transmission have however been observed when these mice are exposed to BSE that has been experimentally transmitted through sheep or goats, indicating that humans may potentially be more susceptible to BSE from small ruminants. Here we demonstrate that increased transmissibility of small ruminant BSE to HuTg mice was not due to replication of higher levels of infectivity in sheep brain tissue, and is instead due to other specific changes in the infectious agent.

  15. Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Yi-zhen; ZHANG Jun; LIU Zeng-li; DU Shou-ying; SHEN Yong-mei

    2010-01-01

    Background The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake.The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting.We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of 131I in a lung cancer cell line in vitro.Methods The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack.The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system.A positive control adenovirusAd-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly.A549 cells were transduced with recombinant adenoviruses.125I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function.Toxic effects of 131I on tumor cells were studied by in vitro clonogenic assay.Results We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter.When infected with recombinant adenovirus constructs expressing hNIS directed by hTERTand CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30- fold compared to the control parental cells, respectively.The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaCIO4).The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P <0.001).Conclusion

  16. Transgenics, agroindustry and food sovereignty

    Directory of Open Access Journals (Sweden)

    Xavier Alejandro León Vega

    2014-10-01

    Full Text Available Food sovereignty has been implemented constitutionally in Ecuador; however, many of the actions and policies are designed to benefit the dominant model of food production, based in agroindustry, intensive monocultures, agrochemicals and transgenics. This article reflects upon the role of family farming as a generator of food sovereignty, and secondly the threat to them by agroindustry agriculture based in transgenic. The role played by food aid in the introduction of transgenic in Latin America and other regions of the world is also analyzed.

  17. Transgenic technologies to induce sterility

    Directory of Open Access Journals (Sweden)

    Wimmer Ernst A

    2009-11-01

    Full Text Available Abstract The last few years have witnessed a considerable expansion in the number of tools available to perform molecular and genetic studies on the genome of Anopheles mosquitoes, the vectors of human malaria. As a consequence, knowledge of aspects of the biology of mosquitoes, such as immunity, reproduction and behaviour, that are relevant to their ability to transmit disease is rapidly increasing, and could be translated into concrete benefits for malaria control strategies. Amongst the most important scientific advances, the development of transgenic technologies for Anopheles mosquitoes provides a crucial opportunity to improve current vector control measures or design novel ones. In particular, the use of genetic modification of the mosquito genome could provide for a more effective deployment of the sterile insect technique (SIT against vector populations in the field. Currently, SIT relies on the release of radiation sterilized males, which compete with wild males for mating with wild females. The induction of sterility in males through the genetic manipulation of the mosquito genome, already achieved in a number of other insect species, could eliminate the need for radiation and increase the efficiency of SIT-based strategies. This paper provides an overview of the mechanisms already in use for inducing sterility by transgenesis in Drosophila and other insects, and speculates on possible ways to apply similar approaches to Anopheles mosquitoes.

  18. Anatomy of the pectoral and forelimb muscles of wildtype and green fluorescent protein-transgenic axolotls and comparison with other tetrapods including humans: a basis for regenerative, evolutionary and developmental studies

    Science.gov (United States)

    Diogo, R; Tanaka, E M

    2012-01-01

    The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers. On the one hand, the inclusion of GFP-transgenic animals allows us to show the muscles as more commonly seen, and thus easier to understand, by current developmental and regenerative biologists. On the other hand, by including wildtype and GFP-transgenic animals and by visualizing these latter animals with and without a simultaneous transmission laser light, we were able to obtain a more complete and clearer understanding of the exact limit of the fleshy and tendinous parts of the muscles and their specific connections with the skeletal elements. This in turn allowed us to settle some controversies in previous anatomical and comparative studies. As most developmental, regenerative and evolutionary biologists are interested in comparing their observations of A. mexicanum with observations in other model organisms, and ultimately in using this information to increase the understanding of human evolution and medicine, we also provide tables showing the homologies between the pectoral and forelimb muscles of axolotls, of model organisms such as mice, frogs and chicken, and of Homo sapiens. An example illustrating the outcomes of using our methodology and of our observations is that they revealed that, contrary to what is often

  19. Anatomy of the pectoral and forelimb muscles of wildtype and green fluorescent protein-transgenic axolotls and comparison with other tetrapods including humans: a basis for regenerative, evolutionary and developmental studies.

    Science.gov (United States)

    Diogo, R; Tanaka, E M

    2012-12-01

    The axolotl Ambystoma mexicanum is one of the most used model organisms in evolutionary, developmental and regenerative studies, particularly because it can reconstitute a fully functional and complete forelimb/hindlimb. Surprisingly, there is no publication that describes all the pectoral and forelimb muscles of this species or provides a comparative framework between these muscles and those of other model organisms and of modern humans. In the present paper we describe and illustrate all these muscles in A. mexicanum and provide the first report about the myology of adults of a model organism that is based on analyses and dissections of both wildtype animals and transgenic animals that express green fluorescent protein (GFP) in muscle fibers. On the one hand, the inclusion of GFP-transgenic animals allows us to show the muscles as more commonly seen, and thus easier to understand, by current developmental and regenerative biologists. On the other hand, by including wildtype and GFP-transgenic animals and by visualizing these latter animals with and without a simultaneous transmission laser light, we were able to obtain a more complete and clearer understanding of the exact limit of the fleshy and tendinous parts of the muscles and their specific connections with the skeletal elements. This in turn allowed us to settle some controversies in previous anatomical and comparative studies. As most developmental, regenerative and evolutionary biologists are interested in comparing their observations of A. mexicanum with observations in other model organisms, and ultimately in using this information to increase the understanding of human evolution and medicine, we also provide tables showing the homologies between the pectoral and forelimb muscles of axolotls, of model organisms such as mice, frogs and chicken, and of Homo sapiens. An example illustrating the outcomes of using our methodology and of our observations is that they revealed that, contrary to what is often

  20. The effects of Fe2O3 nanoparticles on physiology and insecticide activity in non-transgenic and Bt-transgenic cotton

    Directory of Open Access Journals (Sweden)

    Nhan eLe Van

    2016-01-01

    Full Text Available As the demands for nanotechnology and nanoparticle (NP applications in agriculture increase, the ecological risk has drawn more attention because of the unpredictable results of interactions between NPs and transgenic crops. In this study, we investigated the effects of various concentrations of Fe2O3 NPs on Bt-transgenic cotton in comparison with conventional cotton for 10 days. Each treatment was conducted in triplicate, and each experiment was repeated three times. Results demonstrated that Fe2O3 nanoparticles (NPs inhibited the plant height and root length of Bt-transgenic cotton and promoted root hairs and biomass of non-transgenic cotton. Nutrients such as Na and K in Bt-transgenic cotton roots increased, while Zn contents decreased with Fe2O3 NPs. Most hormones in the roots of Bt-transgenic cotton increased at low Fe2O3 NP exposure (100 mg·L−1 but decreased at high concentrations of Fe2O3 NPs (1000 mg·L−1. Fe2O3 NPs increased the Bt-toxin in leaves and roots of Bt-transgenic cotton. Fe2O3 NPs were absorbed into roots, then transported to the shoots of both Bt-transgenic and non-transgenic cottons. The bioaccumulation of Fe2O3 NPs in plants might be a potential risk for agricultural crops and affect the environment and human health.

  1. Real-time immuno-PCR: an approach for detection of trace amounts of transgenic proteins.

    Science.gov (United States)

    Kumar, Rajesh; Sinha, Rajeshwar P

    2014-01-01

    The research on manipulation of crop genomes for transgenic development is continuously increasing due to several benefits. The major concerns linked to the effect of transgenic crops are human health and environment sustainability. To monitor transgenic samples in the food chain, several highly sensitive and specific DNA-based and protein-based detection methods are being used. However, real- time immunio-PCR (RT-IPCR) assay would be able to provide a sensitive detection of trace amounts of transgenic proteins or allergens in the samples and help in monitoring these materials. In the present study, we developed a novel RT-IPCR method to monitor CrylAc transgenic protein in samples with an LOD of 100 pg/mL. The assay may also be useful in the evaluation of functional stability of transgenes inserted in the plant genome.

  2. Constitutive expression of interferon-induced human MxA protein in transgenic tobacco plants does not confer resistance to a variety of RNA viruses

    NARCIS (Netherlands)

    Frese, M.; Prins, M.; Ponten, A.; Goldbach, R.W.; Haller, O.; Zeltz, P.

    2000-01-01

    MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses,including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express

  3. Constitutive expression of interferon-induced human MxA protein in transgenic tobacco plants does not confer resistance to a variety of RNA viruses

    NARCIS (Netherlands)

    Frese, M.; Prins, M.; Ponten, A.; Goldbach, R.W.; Haller, O.; Zeltz, P.

    2000-01-01

    MxA is a key component in the interferon-induced antiviral defense in humans. After viral infections, MxA is rapidly induced and accumulates in the cytoplasm. The multiplication of many RNA viruses,including all bunyaviruses tested so far, is inhibited by MxA. These findings prompted us to express M

  4. Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates

    NARCIS (Netherlands)

    Haji Abdolvahab, Mohadeseh; Fazeli, Ahmad; Halim, Andhyk|info:eu-repo/dai/nl/412608294; Sediq, Ahmad S; Fazeli, Mohammad Reza; Schellekens, Huub|info:eu-repo/dai/nl/068406762

    2016-01-01

    Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNβ-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well

  5. Differential interaction of Apolipoprotein-E isoforms with insulin receptors modulates brain insulin signaling in mutant human amyloid precursor protein transgenic mice.

    Science.gov (United States)

    Chan, Elizabeth S; Chen, Christopher; Cole, Gregory M; Wong, Boon-Seng

    2015-09-08

    It is unclear how human apolipoprotein E4 (ApoE4) increases the risk for Alzheimer's disease (AD). Although Aβ levels can lead to insulin signaling impairment, these experiments were done in the absence of human ApoE. To examine ApoE role, we crossed the human ApoE-targeted replacement mice with mutant human amyloid precursor protein (APP) mice. In 26 week old mice with lower Aβ levels, the expression and phosphorylation of insulin signaling proteins remained comparable among APP, ApoE3xAPP and ApoE4xAPP mouse brains. When the mice aged to 78 weeks, these proteins were markedly reduced in APP and ApoE4xAPP mouse brains. While Aβ can bind to insulin receptor, how ApoE isoforms modulate this interaction remains unknown. Here, we showed that ApoE3 had greater association with insulin receptor as compared to ApoE4, regardless of Aβ42 concentration. In contrast, ApoE4 bound more Aβ42 with increasing peptide levels. Using primary hippocampal neurons, we showed that ApoE3 and ApoE4 neurons are equally sensitive to physiological levels of insulin. However, in the presence of Aβ42, insulin failed to elicit a downstream response only in ApoE4 hippocampal neurons. Taken together, our data show that ApoE genotypes can modulate this Aβ-mediated insulin signaling impairment.

  6. Immunogenicity of Recombinant Human Interferon Beta-1b in Immune-Tolerant Transgenic Mice Corresponds with the Biophysical Characteristics of Aggregates

    NARCIS (Netherlands)

    Haji Abdolvahab, Mohadeseh; Fazeli, Ahmad; Halim, Andhyk; Sediq, Ahmad S; Fazeli, Mohammad Reza; Schellekens, Huub

    2016-01-01

    Determining to what extent biophysical characteristics of aggregates affect immunogenicity of therapeutic interferon beta-1b. Three recombinant human interferon beta-1b (rhIFNβ-1b) samples with different levels of aggregates generated by copper oxidation, thermal stress, or left untreated, as well a

  7. Transgenic mice in developmental toxicology

    Energy Technology Data Exchange (ETDEWEB)

    Woychik, R.P.

    1992-01-01

    Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areas of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.

  8. Transgenic mice in developmental toxicology

    Energy Technology Data Exchange (ETDEWEB)

    Woychik, R.P.

    1992-12-31

    Advances in molecular biology and embryology are being utilized for the generation of transgenic mice, animals that contain specific additions, deletions, or modifications of genes or sequences in their DNA. Mouse embryonic stem cells and homologous recombination procedures have made it possible to target specific DNA structural alterations to highly localized region in the host chromosomes. The majority of the DNA structural rearrangements in transgenic mice can be passed through the germ line and used to establish new genetic traits in the carrier animals. Since the use of transgenic mice is having such an enormous impact on so many areas of mammalian biological research, including developmental toxicology, the objective of this review is to briefly describe the fundamental methodologies for generating transgenic mice and to describe one particular application that has direct relevance to the field of genetic toxicology.

  9. Characterization of atherosclerotic lesions in apo E3-leiden transgenic mice

    NARCIS (Netherlands)

    Leppänen, P.; Luoma, J.S.; Hofker, M.H.; Havekes, L.M.; Ylä-Herttuala, S.

    1998-01-01

    Apo E3-leiden transgenic mice express human dysfunctional apo E variant and develop hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. We characterized diet-induced atherosclerotic lesions in apo E3-leiden transgenic mice using immunocytochemical methods in order to examine foam

  10. Virtual Transgenics: Using a Molecular Biology Simulation to Impact Student Academic Achievement and Attitudes

    Science.gov (United States)

    Shegog, Ross; Lazarus, Melanie M.; Murray, Nancy G.; Diamond, Pamela M.; Sessions, Nathalie; Zsigmond, Eva

    2012-01-01

    The transgenic mouse model is useful for studying the causes and potential cures for human genetic diseases. Exposing high school biology students to laboratory experience in developing transgenic animal models is logistically prohibitive. Computer-based simulation, however, offers this potential in addition to advantages of fidelity and reach.…

  11. Characterization of atherosclerotic lesions in apo E3-leiden transgenic mice

    NARCIS (Netherlands)

    Leppänen, P.; Luoma, J.S.; Hofker, M.H.; Havekes, L.M.; Ylä-Herttuala, S.

    1998-01-01

    Apo E3-leiden transgenic mice express human dysfunctional apo E variant and develop hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. We characterized diet-induced atherosclerotic lesions in apo E3-leiden transgenic mice using immunocytochemical methods in order to examine foam

  12. Heterologous expression in transgenic mosquitoes

    Institute of Scientific and Technical Information of China (English)

    Santhosh P K; Yu hua Deng; Weidong Gu; Xiaoguang Chen

    2010-01-01

    Arthropod-borne diseases such as malaria and dengue virus afflict billions of people worldwide imposing major economic and social burdens. Control of such pathogens is mainly performed by vector management and treatment of affected individuals with drugs. The failure of these conventional approaches due to emergence of insecticide-resistant insects and drug-resistant parasites demonstrate the need of novel and efficacious control strategies to combat these diseases. Genetic modification(GM) of mosquito vectors to impair their ability to be infected and transmit pathogens has emerged as a new strategy to reduce transmission of many vector-borne diseases and deliver public health gains. Several advances in developing transgenic mosquitoes unable to transmit pathogens have gained support, some of them attempt to manipulate the naturally occurring endogenous refractory mechanisms, while others initiate the identification of an exogenous foreign gene which disrupt the pathogen development in insect vectors. Heterologous expression of transgenes under a native or heterologous promoter is important for the screening and effecting of the transgenic mosquitoes. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of this transgenic approach. This review examines these two aspects and describes the basic research work that has been accomplished towards understanding the complex relation between the parasite and its vector and focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to vector-borne disease transmission.

  13. Gene therapy: X-SCID transgene leukaemogenicity.

    Science.gov (United States)

    Thrasher, Adrian J; Gaspar, H Bobby; Baum, Christopher; Modlich, Ute; Schambach, Axel; Candotti, Fabio; Otsu, Makoto; Sorrentino, Brian; Scobie, Linda; Cameron, Ewan; Blyth, Karen; Neil, Jim; Abina, Salima Hacein-Bey; Cavazzana-Calvo, Marina; Fischer, Alain

    2006-09-21

    Gene therapy has been remarkably effective for the immunological reconstitution of patients with severe combined immune deficiency, but the occurrence of leukaemia in a few patients has stimulated debate about the safety of the procedure and the mechanisms of leukaemogenesis. Woods et al. forced high expression of the corrective therapeutic gene IL2RG, which encodes the gamma-chain of the interleukin-2 receptor, in a mouse model of the disease and found that tumours appeared in a proportion of cases. Here we show that transgenic IL2RG does not necessarily have potent intrinsic oncogenic properties, and argue that the interpretation of this observation with respect to human trials is overstated.

  14. The human lactase persistence-associated SNP −13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression

    OpenAIRE

    Fang, Lin; Ahn, Jong Kun; Wodziak, Dariusz; Sibley, Eric

    2012-01-01

    Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in Northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 1...

  15. HuCNS-SC Human NSCs Fail to Differentiate, Form Ectopic Clusters, and Provide No Cognitive Benefits in a Transgenic Model of Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Samuel E. Marsh

    2017-02-01

    Full Text Available Transplantation of neural stem cells (NSCs can improve cognition in animal models of Alzheimer's disease (AD. However, AD is a protracted disorder, and prior studies have examined only short-term effects. We therefore used an immune-deficient model of AD (Rag-5xfAD mice to examine long-term transplantation of human NSCs (StemCells Inc.; HuCNS-SCs. Five months after transplantation, HuCNS-SCs had engrafted and migrated throughout the hippocampus and exhibited no differences in survival or migration in response to β-amyloid pathology. Despite robust engraftment, HuCNS-SCs failed to terminally differentiate and over a quarter of the animals exhibited ectopic human cell clusters within the lateral ventricle. Unlike prior short-term experiments with research-grade HuCNS-SCs, we also found no evidence of improved cognition, no changes in brain-derived neurotrophic factor, and no increase in synaptic density. These data, while disappointing, reinforce the notion that individual human NSC lines need to be carefully assessed for efficacy and safety in appropriate long-term models.

  16. Single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk.

    Science.gov (United States)

    Tillib, S V; Privezentseva, M E; Ivanova, T I; Vasilev, L F; Efimov, G A; Gursky, Y G; Georgiev, G P; Goldman, I L; Sadchikova, E R

    2014-02-15

    Single-domain antibody generation technology was applied to make new Sepharose-bound ligands for affinity separation of closely related proteins, such as human and goat lactoferrin. We generated recombinant antibodies that can selectively bind/recognize only lactoferrins having amino acid sequences identical to that of human natural lactoferrin (anti-hLF Ab). Selected and purified histidine-tagged single-domain antibodies were used as ligands, and different lactoferrins were used as analytes in the kinetics analysis of lactoferrin binding to captured anti-hLF Abs using the Bio-Rad ProteOn XPR36 protein interaction array system. The data obtained were consistent with a 1:1 binding model with very high affinity, practically equal in the case of hLF and rec-hLF (calculated KD varied from 0.43nM to 3.7nM). Interaction of captured fsdAbs with goat LF was significantly weaker and not detectable under the same analysis conditions. We demonstrated the high efficiency of the recombinant human lactoferrin purification from goat lactoferrin and other proteins using the obtained single domain antibody-based affinity ligands. We believe this approach can be used for the generation of single-domain antibody-based affinity media for the efficient separation/purification of a wide spectrum of other highly homologous proteins.

  17. Heart-specific expression of laminopathic mutations in transgenic zebrafish.

    Science.gov (United States)

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

    Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

  18. Longitudinal testing of hippocampal plasticity reveals the onset and maintenance of endogenous human Aß-induced synaptic dysfunction in individual freely behaving pre-plaque transgenic rats: rapid reversal by anti-Aß agents.

    Science.gov (United States)

    Qi, Yingjie; Klyubin, Igor; Harney, Sarah C; Hu, NengWei; Cullen, William K; Grant, Marianne K; Steffen, Julia; Wilson, Edward N; Do Carmo, Sonia; Remy, Stefan; Fuhrmann, Martin; Ashe, Karen H; Cuello, A Claudio; Rowan, Michael J

    2014-12-24

    Long before synaptic loss occurs in Alzheimer's disease significant harbingers of disease may be detected at the functional level. Here we examined if synaptic long-term potentiation is selectively disrupted prior to extracellular deposition of Aß in a very complete model of Alzheimer's disease amyloidosis, the McGill-R-Thy1-APP transgenic rat. Longitudinal studies in freely behaving animals revealed an age-dependent, relatively rapid-onset and persistent inhibition of long-term potentiation without a change in baseline synaptic transmission in the CA1 area of the hippocampus. Thus the ability of a standard 200 Hz conditioning protocol to induce significant NMDA receptor-dependent short- and long-term potentiation was lost at about 3.5 months of age and this deficit persisted for at least another 2-3 months, when plaques start to appear. Consistent with in vitro evidence for a causal role of a selective reduction in NMDA receptor-mediated synaptic currents, the deficit in synaptic plasticity in vivo was associated with a reduction in the synaptic burst response to the conditioning stimulation and was overcome using stronger 400 Hz stimulation. Moreover, intracerebroventricular treatment for 3 days with an N-terminally directed monoclonal anti- human Aß antibody, McSA1, transiently reversed the impairment of synaptic plasticity. Similar brief treatment with the BACE1 inhibitor LY2886721 or the γ-secretase inhibitor MRK-560 was found to have a comparable short-lived ameliorative effect when tracked in individual rats. These findings provide strong evidence that endogenously generated human Aß selectively disrupts the induction of long-term potentiation in a manner that enables potential therapeutic options to be assessed longitudinally at the pre-plaque stage of Alzheimer's disease amyloidosis.

  19. Human neural stem cells improve cognition and promote synaptic growth in two complementary transgenic models of Alzheimer's disease and neuronal loss.

    Science.gov (United States)

    Ager, Rahasson R; Davis, Joy L; Agazaryan, Andy; Benavente, Francisca; Poon, Wayne W; LaFerla, Frank M; Blurton-Jones, Mathew

    2015-07-01

    Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder, affecting over 35 million people worldwide. Pathologically, AD is characterized by the progressive accumulation of β-amyloid (Aβ) plaques and neurofibrillary tangles within the brain. Together, these pathologies lead to marked neuronal and synaptic loss and corresponding impairments in cognition. Current treatments, and recent clinical trials, have failed to modify the clinical course of AD; thus, the development of novel and innovative therapies is urgently needed. Over the last decade, the potential use of stem cells to treat cognitive impairment has received growing attention. Specifically, neural stem cell transplantation as a treatment for AD offers a novel approach with tremendous therapeutic potential. We previously reported that intrahippocampal transplantation of murine neural stem cells (mNSCs) can enhance synaptogenesis and improve cognition in 3xTg-AD mice and the CaM/Tet-DT(A) model of hippocampal neuronal loss. These promising findings prompted us to examine a human neural stem cell population, HuCNS-SC, which has already been clinically tested for other neurodegenerative disorders. In this study, we provide the first evidence that transplantation of research grade HuCNS-SCs can improve cognition in two complementary models of neurodegeneration. We also demonstrate that HuCNS-SC cells can migrate and differentiate into immature neurons and glia and significantly increase synaptic and growth-associated markers in both 3xTg-AD and CaM/Tet-DTA mice. Interestingly, improvements in aged 3xTg-AD mice were not associated with altered Aβ or tau pathology. Rather, our findings suggest that human NSC transplantation improves cognition by enhancing endogenous synaptogenesis. Taken together, our data provide the first preclinical evidence that human NSC transplantation could be a safe and effective therapeutic approach for treating AD. © 2014 The Authors. Hippocampus

  20. A haplotype of human angiotensinogen gene containing −217A increases blood pressure in transgenic mice compared with −217G

    OpenAIRE

    Jain, Sudhir; Vinukonda, Govindaiah; Fiering, Steven N; Kumar, Ashok

    2008-01-01

    The human angiotensinogen (hAGT) gene contains an A/G polymorphism at −217, and frequency of −217A allele is increased in African-American hypertensive patients. The hAGT gene has seven polymorphic sites in the 1.2-kb region of its promoter, and variant −217A almost always occurs with −532T, −793A, and −1074T, whereas variant −217G almost always occurs with −532C, −793G, and −1074G. Since allele −6A is the predominant allele in African-Americans, the AGT gene can be subdivided into two main h...

  1. Genetic treatment of severe hemoglobinopathies: the combat against transgene variegation and transgene silencing.

    Science.gov (United States)

    Rivella, S; Sadelain, M

    1998-04-01

    Gene addition strategies are rational approaches to the treatment of sickle cell anemia and thalassemia. The goal of such genetic treatments is to introduce a functional globin transcription unit in hematopoietic stem cells and express the transgene in a manner that is erythroid-specific, elevated, relatively constant from one cell to another, and sustained over time. Gene transfer is mediated by an expanding array of viral and nonviral vectors. High-titer retroviral vectors harboring the human beta-globin gene and the core sequences of the human beta-globin locus control region yield erythroid-specific gene expression in erythroid cell lines and in short-term murine bone marrow chimeras. However, we show that expression remains subject to position effect variegation and often decreases over time in vivo. Rather than a progressive transcriptional silencing in all cells, we ascribe the waning expression to the gradual emergence in blood of erythroid progeny derived from more and more primitive precursor cells in the months after transplantation. In our model, transgene expression is therefore determined by the integration site and the differentiation stage of the transduced cell at the time of integration. Globin expression is thus different in the progeny of a transduced erythroid progenitor cell and in the erythroid progeny of a transduced hematopoietic stem cell, reflecting the effect of flanking chromatin in differentiated cells and of chromatin remodeling at the site of integration in the progeny of multipotential cells. This model predicts that insulators and matrix attachment regions could be highly valuable to gene therapy in combination with potent transcriptional activators. When efficient gene transfer in hematopoietic stem cells is achieved at last, the challenge will be to regulate gene expression in vivo and overcome transgene variegation and transgene silencing.

  2. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco.

    Science.gov (United States)

    Higo, K; Saito, Y; Higo, H

    1993-09-01

    Nicotiana tabacum was transformed with a chemically synthesized gene encoding the human epidermal growth factor (hEGF) under control of the CaMV-35S promoter. The hEGF gene sequence was present at one to several copies in the primary transformant plants (R0), and a transcript with the expected length was produced. Slot blot analysis of total RNAs of the progeny (R1) seedlings, originating from self-pollination of the R0 plants, showed that the level of mRNA expression was generally, but not always, heritable. The highest hEGF peptide content per unit of total soluble protein in young (upper) R1 leaves so far examined by an immunological method was about 0.001%. These results suggest that either the hEGF peptide was less stable than the average leaf protein, or the hEGF mRNAs were not efficiently translated.

  3. TRANSGENIC PLANTS RESISTANT TO INSECTS

    Directory of Open Access Journals (Sweden)

    S. Kereša

    2009-09-01

    Full Text Available Proteinase inhibitors are secondary metabolites present in all plants and it seems that their major role is protection of plants against attacks of animals, insects and microorganisms. One of the family of proteinase inhibitors are squash inhibitors of serine proteinases purified from seeds belonging to genera Cucurbita, Cucumis and Momordica. Squash inhibitors consist of 29-32 amino acid residues and are considered to be the smallest inhibitors of the serine proteinases known. Because of shortness, genes for these inhibitors could be synthesised and modified at different ways. Modifications could lead to changes in inhibitor activity. Tobacco as a model plant was transformed with 12 different genes of squash inhibitors. Stable integration of transgenes in putative transgenic plants was determined by PCR analysis using genomic DNA and primers that anneal to promoter and terminator region. The first step of proteinase inhibitor gene expression in transgenic plants was revealed by RT-PCR analysis. In entomological tests where larvae were fed with leaves, influence of transgenic T0 plants, as well as non-transgenic control plants on retardation of larval growth of S. littoralis was examined. Results of entomological tests showed that it is possible to express squash proteinase inhibitors in plants at level that significantly reduces S. littoralis larval growth.

  4. Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T cells specifically inhibit Ewing sarcoma growth in vitro and in vivo.

    Science.gov (United States)

    Blaeschke, Franziska; Thiel, Uwe; Kirschner, Andreas; Thiede, Melanie; Rubio, Rebeca Alba; Schirmer, David; Kirchner, Thomas; Richter, Günther H S; Mall, Sabine; Klar, Richard; Riddell, Stanley; Busch, Dirk H; Krackhardt, Angela; Grunewald, Thomas G P; Burdach, Stefan

    2016-07-12

    The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion.In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction.After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2-/-É£c-/- mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells.CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic.

  5. Generation of multipotent early lymphoid progenitors from human embryonic stem cells.

    Science.gov (United States)

    Larbi, Aniya; Mitjavila-Garcia, Maria Teresa; Flamant, Stéphane; Valogne, Yannick; Clay, Denis; Usunier, Benoît; l'Homme, Bruno; Féraud, Olivier; Casal, Ibrahim; Gobbo, Emilie; Divers, Dominique; Chapel, Alain; Turhan, Ali G; Bennaceur-Griscelli, Annelise; Haddad, Rima

    2014-12-15

    During human embryonic stem cell (ESC) hematopoietic differentiation, the description of the initial steps of lymphopoiesis remains elusive. Using a two-step culture procedure, we identified two original populations of ESC-derived hematopoietic progenitor cells (HPCs) with CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) phenotypes. Bulk cultures and limiting dilution assays, culture with MS5 cells in the presence of Notch ligand Delta-like-1 (DL-1), and ex vivo colonization tests using fetal thymic organ cultures showed that although CD34(+)CD45RA(+)CD7(-) HPCs could generate cells of the three lymphoid lineages, their potential was skewed toward the B cell lineages. In contrast, CD34(+)CD45RA(+)CD7(+) HPCs predominantly exhibited a T/natural killer (NK) cell differentiation potential. Furthermore these cells could differentiate equivalently into cells of the granulo-macrophagic lineage and dendritic cells and lacked erythroid potential. Expression profiling of 18 markers by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs express genes of the lymphoid specification and that CD34(+)CD45RA(+)CD7(-) cells express B-cell-associated genes, while CD34(+)CD45RA(+)CD7(+) HPCs display a T-cell molecular profile. Altogether, these findings indicate that CD34(+)CD45RA(+)CD7(-) and CD34(+)CD45RA(+)CD7(+) HPCs correspond to candidate multipotent early lymphoid progenitors polarized toward either the B or T/NK lineage, respectively. This work should improve our understanding of the early steps of lymphopoiesis from pluripotent stem cells and pave the way for the production of lymphocytes for cell-based immunotherapy and lymphoid development studies.

  6. Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

    Energy Technology Data Exchange (ETDEWEB)

    Chakravarthy, Balu, E-mail: Balu.Chakravarthy@nrc-cnrc.gc.ca [Human Health Therapeutics, National Research Council of Canada, Ottawa, ON (Canada); Gaudet, Chantal; Menard, Michel; Brown, Leslie; Atkinson, Trevor [Human Health Therapeutics, National Research Council of Canada, Ottawa, ON (Canada); LaFerla, Frank M. [Department of Neurobiology and Behavior, University of California, Irvine, CA (United States); Ito, Shingo [Human Health Therapeutics, National Research Council of Canada, Ottawa, ON (Canada); Armato, Ubaldo; Dal Pra, Ilaria [Department of Life and Reproduction Sciences, University of Verona Medical School, Verona (Italy); Whitfield, James [Human Health Therapeutics, National Research Council of Canada, Ottawa, ON (Canada)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatin receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.

  7. Melissa officinalis essential oil reduces plasma triglycerides in human apolipoprotein E2 transgenic mice by inhibiting sterol regulatory element-binding protein-1c-dependent fatty acid synthesis.

    Science.gov (United States)

    Jun, Hee-Jin; Lee, Ji Hae; Jia, Yaoyao; Hoang, Minh-Hien; Byun, Hanna; Kim, Kyoung Heon; Lee, Sung-Joon

    2012-03-01

    We investigated the hypolipidemic effects of Melissa officinalis essential oil (MOEO) in human APOE2 transgenic mice and lipid-loaded HepG2 cells. Plasma TG concentrations were significantly less in APOE2 mice orally administered MOEO (12.5 μg/d) for 2 wk than in the vehicle-treated group. Cellular TG and cholesterol concentrations were also significantly decreased in a dose- (400 and 800 mg/L) and time- (12 and 24 h) dependent manner in HepG2 cells stimulated with MOEO compared with controls. Mouse hepatic transcriptome analysis suggested MOEO feeding altered several lipid metabolic pathways, including bile acid and cholesterol synthesis and fatty acid metabolism. In HepG2 cells, the rate of fatty acid oxidation, as assessed using [1-(14)C]palmitate, was unaltered; however, the rate of fatty acid synthesis quantified with [1-(14)C]acetate was significantly reduced by treatment with 400 and 800 mg/L MOEO compared with untreated controls. This reduction was due to the decreased expression of SREBP-1c and its responsive genes in fatty acid synthesis, including FAS, SCD1, and ACC1. Subsequent chromatin immunoprecipitation analysis further demonstrated that the binding of p300/CBP-associated factor, a coactivator of SREBP-1c, and histone H3 lysine 14 acetylation at the FAS, SCD1, and ACC1 promoters were significantly reduced in the livers of APOE2 mice and HepG2 cells treated with MOEO compared with their controls. Additionally, MOEO stimulation in HepG2 cells induced bile acid synthesis and reduced the nuclear form of SREBP-2, a key transcription factor in hepatic cholesterol synthesis. These findings suggest that the intake of phytochemicals with pleasant scent could have beneficial metabolic effects.

  8. A Herpes Simplex Virus Type 1 Human Asymptomatic CD8+ T-Cell Epitopes-Based Vaccine Protects Against Ocular Herpes in a “Humanized” HLA Transgenic Rabbit Model

    Science.gov (United States)

    Srivastava, Ruchi; Khan, Arif A.; Huang, Jiawei; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

    2015-01-01

    Purpose. A clinical vaccine that protects from ocular herpes simplex virus type 1 (HSV-1) infection and disease still is lacking. In the present study, preclinical vaccine trials of nine asymptomatic (ASYMP) peptides, selected from HSV-1 glycoproteins B (gB), and tegument proteins VP11/12 and VP13/14, were performed in the “humanized” HLA–transgenic rabbit (HLA-Tg rabbit) model of ocular herpes. We recently reported that these peptides are highly recognized by CD8+ T cells from “naturally” protected HSV-1–seropositive healthy ASYMP individuals (who have never had clinical herpes disease). Methods. Mixtures of three ASYMP CD8+ T-cell peptides derived from either HSV-1 gB, VP11/12, or VP13/14 were delivered subcutaneously to different groups of HLA-Tg rabbits (n = 10) in incomplete Freund's adjuvant, twice at 15-day intervals. The frequency and function of HSV-1 epitope-specific CD8+ T cells induced by these peptides and their protective efficacy, in terms of survival, virus replication in the eye, and ocular herpetic disease were assessed after an ocular challenge with HSV-1 (strain McKrae). Results. All mixtures elicited strong and polyfunctional IFN-γ– and TNF-α–producing CD107+CD8+ cytotoxic T cells, associated with a significant reduction in death, ocular herpes infection, and disease (P herpes, and provide a prototype vaccine formulation that may be highly efficacious for preventing ocular herpes in humans. PMID:26098469

  9. Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi.

    Directory of Open Access Journals (Sweden)

    Alison T Isaacs

    2011-04-01

    Full Text Available Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.

  10. CCK Response Deficiency in Synphilin-1 Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Wanli W Smith

    Full Text Available Previously, we have identified a novel role for the cytoplasmic protein, synphilin-1(SP1, in the controls of food intake and body weight in both mice and Drosophila. Ubiquitous overexpression of human SP1 in brain neurons in transgenic mice results in hyperphagia expressed as an increase in meal size. However, the mechanisms underlying this action of SP1 remain to be determined. Here we investigate a potential role for altered gut feedback signaling in the effects of SP1 on food intake. We examined responses to peripheral administration of cholecytokinin (CCK, amylin, and the glucagon like peptide-1 (GLP-1 receptor agonist, exendin-4. Intraperitoneal administration of CCK at doses ranging from 1-10 nmol/kg significantly reduced glucose intake in wild type (WT mice, but failed to affect intake in SP1 transgenic mice. Moreover, there was a significant attenuation of CCK-induced c-Fos expression in the dorsal vagal complex in SP1 transgenic mice. In contrast, WT and SP1 transgenic mice were similarly responsive to both amylin and exendin-4 treatment. These studies demonstrate that SP1 results in a CCK response deficiency that may contribute to the increased meal size and overall hyperphagia in synphillin-1 transgenic mice.

  11. CCK Response Deficiency in Synphilin-1 Transgenic Mice.

    Science.gov (United States)

    Smith, Wanli W; Smith, Megan; Yang, Dejun; Choi, Pique P; Moghadam, Alexander; Li, Tianxia; Moran, Timothy H

    2015-01-01

    Previously, we have identified a novel role for the cytoplasmic protein, synphilin-1(SP1), in the controls of food intake and body weight in both mice and Drosophila. Ubiquitous overexpression of human SP1 in brain neurons in transgenic mice results in hyperphagia expressed as an increase in meal size. However, the mechanisms underlying this action of SP1 remain to be determined. Here we investigate a potential role for altered gut feedback signaling in the effects of SP1 on food intake. We examined responses to peripheral administration of cholecytokinin (CCK), amylin, and the glucagon like peptide-1 (GLP-1) receptor agonist, exendin-4. Intraperitoneal administration of CCK at doses ranging from 1-10 nmol/kg significantly reduced glucose intake in wild type (WT) mice, but failed to affect intake in SP1 transgenic mice. Moreover, there was a significant attenuation of CCK-induced c-Fos expression in the dorsal vagal complex in SP1 transgenic mice. In contrast, WT and SP1 transgenic mice were similarly responsive to both amylin and exendin-4 treatment. These studies demonstrate that SP1 results in a CCK response deficiency that may contribute to the increased meal size and overall hyperphagia in synphillin-1 transgenic mice.

  12. Generation of the regulatory protein rtTA transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Kang Xu; Xin-Yan Deng; Ying Yue; Zhong-Min Guo; Bing Huang; Xun Hong; Dong Xiao; Xi-Gu Chen

    2005-01-01

    AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome "immune tolerance" formed during the embryonic development and "immune escape" against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

  13. Application of a nuclear localization signal gene in transgene mice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Efficient gene transfer by cytoplasm co-injec- tion will offer a powerful means for transgenic animals. Using co-injection in cytoplasm, two independent gene constructs, including bovine (?-s1-casein-hG-CSF and a mammal expression vector expressing a nuclear localization signal (mNLS), were introduced into fertilized mouse eggs. The target gene construct was docked into host nucleus probably by the nuclear localization signal. Transgene mice have been obtained at 58% (29/50) of integration ratio. Expression level of the positive transgene mice was detected by Western blotting. Maximal expression of human G-CSF was estimated about 540 mg/L of milk. The expression ratio was up to 75% (9/12). The results here have important practical implications for the generation of mammary gland bioreactors and other transgene studies. Co-injection of a target gene with an expression vector of a mammal nuclear localization signal by cytoplasm appears to be a useful, efficient and easy strategy for generating transgenic animals, which may be able to substitute the routine method of pronucleus-injection of fertilized eggs.

  14. Metabolic disruption identified in the Huntington's disease transgenic sheep model.

    Science.gov (United States)

    Handley, Renee R; Reid, Suzanne J; Patassini, Stefano; Rudiger, Skye R; Obolonkin, Vladimir; McLaughlan, Clive J; Jacobsen, Jessie C; Gusella, James F; MacDonald, Marcy E; Waldvogel, Henry J; Bawden, C Simon; Faull, Richard L M; Snell, Russell G

    2016-02-11

    Huntington's disease (HD) is a dominantly inherited, progressive neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of HTT, encoding huntingtin. There are no therapies that can delay the progression of this devastating disease. One feature of HD that may play a critical role in its pathogenesis is metabolic disruption. Consequently, we undertook a comparative study of metabolites in our transgenic sheep model of HD (OVT73). This model does not display overt symptoms of HD but has circadian rhythm alterations and molecular changes characteristic of the early phase disease. Quantitative metabolite profiles were generated from the motor cortex, hippocampus, cerebellum and liver tissue of 5 year old transgenic sheep and matched controls by gas chromatography-mass spectrometry. Differentially abundant metabolites were evident in the cerebellum and liver. There was striking tissue-specificity, with predominantly amino acids affected in the transgenic cerebellum and fatty acids in the transgenic liver, which together may indicate a hyper-metabolic state. Furthermore, there were more strong pair-wise correlations of metabolite abundance in transgenic than in wild-type cerebellum and liver, suggesting altered metabolic constraints. Together these differences indicate a metabolic disruption in the sheep model of HD and could provide insight into the presymptomatic human disease.

  15. Transgenic woody plants for biofuel

    Institute of Scientific and Technical Information of China (English)

    Wei Tang; Anna Y.Tang

    2014-01-01

    Transgenic trees as a new source for biofuel have brought a great interest in tree biotechnology. Genetically modifying forest trees for ethanol production have advantages in technical challenges, costs, environmental concerns, and financial problems over some of crops. Genetic engineering of forest trees can be used to reduce the level of lignin, to produce the fast-growing trees, to develop trees with higher cellulose, and to allow the trees to be grown more widely. Trees can establish themselves in the field with less care of farmers, compared to most of crops. Transgenic crops as a new source for biofuel have been recently reviewed in several reviews. Here, we overview transgenic woody plants as a new source for biofuel including genetically modified woody plants and environment; main focus of woody plants genetic modifications;solar to chemical energy transfer; cellulose biosynthesis;lignin biosynthesis;and cellulosic ethanol as biofuel.

  16. A Transgenic Mouse Model of Poliomyelitis.

    Science.gov (United States)

    Koike, Satoshi; Nagata, Noriyo

    2016-01-01

    Transgenic mice (tg mice) that express the human poliovirus receptor (PVR), CD155, are susceptible to poliovirus and develop a neurological disease that resembles human poliomyelitis. Assessment of the neurovirulence levels of poliovirus strains, including mutant viruses produced by reverse genetics, circulating vaccine-derived poliovirus, and vaccine candidates, is useful for basic research of poliovirus pathogenicity, the surveillance of circulating polioviruses, and the quality control of oral live poliovirus vaccines, and does not require the use of monkeys. Furthermore, PVR-tg mice are useful for studying poliovirus tissue tropism and host immune responses. PVR-tg mice can be bred with mice deficient in the genes involved in viral pathogenicity. This report describes the methods used to analyze the pathogenicity and immune responses of poliovirus using the PVR-tg mouse model.

  17. Towards Transgenic Primates:What can we learn from mouse genetics?

    Institute of Scientific and Technical Information of China (English)

    WANG; Phillip; L.; TSIEN; Joe; Z.

    2009-01-01

    Considering the great physiological and behavioral similarities with humans,monkeys represent the ideal models not only for the study of complex cognitive behavior but also for the preclinical research and development of novel therapeutics for treating human diseases.Various powerful genetic tech-nologies initially developed for making mouse models are being explored for generating transgenic primate models.We review the latest genetic engineering technologies and discuss the potentials and limitations for systematic production of transgenic primates.

  18. The methods to generate transgenic animals and to control transgene expression.

    Science.gov (United States)

    Houdebine, Louis-Marie

    2002-09-25

    Transgenic animals have been used for years to study gene function and to create models for the study of human diseases. This approach has become still more justified after the complete sequencing of several genomes. Transgenic animals are ready to become industrial bioreactors for the preparation of pharmaceuticals in milk and probably in the future in egg white. Improvement of animal production by transgenesis is still in infancy. Despite its intensive use, animal transgenesis is still suffering from technical limitations. The generation of transgenics has recently become easier or possible for different species thanks to the use of transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by intracellular sperm injection or not and to the use of the cloning technique using somatic cells in which genes have been added or inactivated. The Cre-LoxP system is more and more used to withdraw a given sequence from the genome or to target the integration of a foreign DNA. The tetracycline system has been improved and can more and more frequently be used to obtain faithful expression of transgenes. Several tools: RNA forming a triple helix with DNA, antisense RNA including double strand RNA inducing RNA interference and ribozymes, and also expression of proteins having a negative transdominant effect, are tentatively being improved to inhibit specifically the expression of host or viral genes.All these techniques are expected to offer experimenters new and more precise models to study gene function even in large animals. Improvement of breeding by transgenesis has become more plausible including through the precise allele replacement in farm animals.

  19. Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-beta1 transgenic mice.

    OpenAIRE

    Clouthier, D.E; Comerford, S A; Hammer, R. E.

    1997-01-01

    Transgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was local...

  20. Transgene landbouwhuisdieren : het overwegen waard?

    NARCIS (Netherlands)

    Linskens, M.

    1989-01-01

    Het rapport geeft informatie over de ontwikkelingen die, momenteel nog vooral in het onderzoek, op dit terrein gaande zijn. Het geeft aan wanneer de eerste transgene landbouwhuisdieren, bij een ongewijzigd beleid, op de boerderij kunnen rondlopen. Verder wordt er inzicht verschaft in de maatschappel

  1. [Biofuels, food security and transgenic crops].

    Science.gov (United States)

    Acosta, Orlando; Chaparro-Giraldo, Alejandro

    2009-01-01

    Soaring global food prices are threatening to push more poor people back below the poverty line; this will probably become aggravated by the serious challenge that increasing population and climate changes are posing for food security. There is growing evidence that human activities involving fossil fuel consumption and land use are contributing to greenhouse gas emissions and consequently changing the climate worldwide. The finite nature of fossil fuel reserves is causing concern about energy security and there is a growing interest in the use of renewable energy sources such as biofuels. There is growing concern regarding the fact that biofuels are currently produced from food crops, thereby leading to an undesirable competition for their use as food and feed. Nevertheless, biofuels can be produced from other feedstocks such as lingo-cellulose from perennial grasses, forestry and vegetable waste. Biofuel energy content should not be exceeded by that of the fossil fuel invested in its production to ensure that it is energetically sustainable; however, biofuels must also be economically competitive and environmentally acceptable. Climate change and biofuels are challenging FAO efforts aimed at eradicating hunger worldwide by the next decade. Given that current crops used in biofuel production have not been domesticated for this purpose, transgenic technology can offer an enormous contribution towards improving biofuel crops' environmental and economic performance. The present paper critically presents some relevant relationships between biofuels, food security and transgenic plant technology.

  2. Modeling Alzheimer's disease in transgenic rats.

    Science.gov (United States)

    Do Carmo, Sonia; Cuello, A Claudio

    2013-10-25

    Alzheimer's disease (AD) is the most common form of dementia. At the diagnostic stage, the AD brain is characterized by the accumulation of extracellular amyloid plaques, intracellular neurofibrillary tangles and neuronal loss. Despite the large variety of therapeutic approaches, this condition remains incurable, since at the time of clinical diagnosis, the brain has already suffered irreversible and extensive damage. In recent years, it has become evident that AD starts decades prior to its clinical presentation. In this regard, transgenic animal models can shed much light on the mechanisms underlying this "pre-clinical" stage, enabling the identification and validation of new therapeutic targets. This paper summarizes the formidable efforts to create models mimicking the various aspects of AD pathology in the rat. Transgenic rat models offer distinctive advantages over mice. Rats are physiologically, genetically and morphologically closer to humans. More importantly, the rat has a well-characterized, rich behavioral display. Consequently, rat models of AD should allow a more sophisticated and accurate assessment of the impact of pathology and novel therapeutics on cognitive outcomes.

  3. Size matters: use of YACs, BACs and PACs in transgenic animals.

    Science.gov (United States)

    Giraldo, P; Montoliu, L

    2001-04-01

    In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.

  4. Pregnancies Resulting from Transgenic Embryos with Human Lactoferrin Gene Produced by Somatic Cell Nuclear Transfer%体细胞核移植法获得转人乳铁蛋白基因克隆山羊妊娠的研究

    Institute of Scientific and Technical Information of China (English)

    刘凤军; 张玉玲; 杨自军; 陈兴启; 孙达权; 王国华; 张涌

    2008-01-01

    [Objective] The aim of this study is to understand the effects of donor cell type, embryo stage, number and transfer position on the efficiency of goat transgenic clone. [Method] Using somatic cell nuclear transfer technology, the single goat fetal fibroblasts (GFF) and mammary gland epithelial cells (GMGE) harboring human lactoferrin (hLF) gene were transferred to the enucleated oocyte. Reconstructed karyoplast-cytoplast couplets were fused, activated, and cultured in vitro. Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients, and blastocysts were transferred into uterine horn. [Result] The pregnancy rate was similar between GFF and GMGE (oviduct transfer:26.47% vs. 20.00%), and between oviduct transfer and uterine horn transfer (26.47% vs. 25.00%) for GFF group; pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group (40.00% vs. 26.67% and 21.43%). [Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type, embryo stage and transfer position but be done by the number of embryo transferred per recipient. In addition, the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.

  5. Functional screening of an asthma QTL in YAC transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Symula, Derek J.; Frazer, Kelly A.; Ueda, Yukihiko; Denefle, Patrice; Stevens, Mary E.; Wang, Zhi-En; Locksley, Richard; Rubin, Edward M.

    1999-07-02

    While large numbers of quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. While large numbers of quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q33, the authors characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a one megabase interva2048 chromosome 5q31 containing 23 genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180 kb region containing 5 genes, including human interleukin 4 (IL4) and interleukin 13 (IL13), which induce IgE class switching in B cells5. Further analysis of these mice and mice transgenic for only murine Il4 and Il13 demonstrated that moderate changes in murine Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled them to sift through multiple genes in the 5q3 asthma QTL without prior consideration of assumed individual gene function and identify genes that influence the QTL phenotype in vivo.

  6. Macrophage specific overexpression of the human macrophage scavenger receptor in transgenic mice, using a 180-kb yeast artificial chromosome, leads to enhanced foam cell formation of isolated peritoneal macrophages

    NARCIS (Netherlands)

    Winther, M.P.J. de; Dijk, K.W. van; Vlijmen, B.J.M. van; Gijbels, M.J.J.; Heus, J.J.; Wijers, E.R.; Bos, A.C. van den; Breuer, M.; Frants, R.R.; Havekes, L.M.; Hofker, M.H.

    1999-01-01

    Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic

  7. Functional and safety evaluation of transgenic pork rich in omega-3 fatty acids.

    Science.gov (United States)

    Tang, Maoxue; Qian, Lili; Jiang, Shengwang; Zhang, Jian; Song, Pengkun; Chen, Yaoxing; Cui, Wentao; Li, Kui

    2014-08-01

    Genetically modified animals rich in omega-3 unsaturated fatty acid offer a new strategy to improve the human health, but at the same time present a challenge in terms of food safety assessment. In this study, we evaluated the function and safety of sFat-1 transgenic pork rich in omega-3 fatty acids in mice by feeding basic diet and diets that contain wild type pork and sFat-1 transgenic pork. Blood biochemistry, haematology, peripheral T cell distributions, bacterial counts, gross necropsy, histopathology and organ weights were performed in mice fed with different doses of wild type and transgenic pork. Results indicated that both low and high dose of wild type and transgenic pork had no significant effect on blood biochemistry, T cell distribution, immunoglobulins and bacterial counts in intestine and feces. However, it was noted that both low and high dose of transgenic pork improved the liver immune system in mice, which is probably due to the beneficial contribution of high level of the "good" fatty acids in transgenic pork. There is no significant effect of transgenic pork on all other organs in mice. In summary, our study clearly demonstrated that feeding transgenic pork rich in omega-3 fatty acids did not cause any harm to mice, and in fact, improved the liver immune system.

  8. A double built-in containment strategy for production of recombinant proteins in transgenic rice.

    Science.gov (United States)

    Zhang, Xianwen; Wang, Dongfang; Zhao, Sinan; Shen, Zhicheng

    2014-01-01

    Using transgenic rice as a bioreactor for mass production of pharmaceutical proteins could potentially reduce the cost of production significantly. However, a major concern over the bioreactor transgenic rice is the risk of its unintended spreading into environment and into food or feed supplies. Here we report a mitigating method to prevent unwanted transgenic rice spreading by a double built-in containment strategy, which sets a selectively termination method and a visual tag technology in the T-DNA for transformation. We created transgenic rice with an inserted T-DNA that harbors a human proinsulin gene fused with the far-red fluorescent protein gene mKate_S158A, an RNAi cassette suppressing the expression of the rice bentazon detoxification enzyme CYP81A6, and an EPSPS gene as the selection marker for transformation. Herbicide spray tests indicated that such transgenic rice plants can be killed selectively by a spray of bentazon at regular field application dosage for rice weed control. Moreover, the transgenic rice seeds were bright red in color due to the fused far-red fluorescent protein, and could be easily visualized under daylight by naked eyes. Thus, the transgenic rice plants reported in this study could be selectively killed by a commonly used herbicide during their growth stage, and their seeds may be detected visually during processing and consumption after harvest. This double built-in containment strategy may greatly enhance the confinement of the transgenic rice.

  9. Development of Hyperplasias, Preneoplasias, and Mammary Tumors in MMTV-c-erbB-2 and MMTV-TGFα Transgenic Rats

    Science.gov (United States)

    Davies, Barry R.; Platt-Higgins, Angela M.; Schmidt, Gunter; Rudland, Philip S.

    1999-01-01

    Human cDNAs corresponding to two epidermal growth factor-related products that are overexpressed in human breast cancers, that for c-erbB-2 (HER-2) and for transforming growth factor α (TGFα), have been cloned downstream of the mouse mammary tumor virus (MMTV) long terminal repeat promoter and injected into the pronucleus of fertilized oocytes of Sprague-Dawley rats to produce transgenic offspring. Expression of the transgenic mRNAs is not detectable in mammary tissue from virgin transgenic rats but is detected in mammary tissue from certain lines of mid-pregnant transgenic rats. When two such lines of either type of transgenic rat are subjected to repeated cycles of pregnancy and lactation, they produce, primarily in the mammary glands, extensive pathologies, whereas virgin transgenic rats produce no such abnormalities. Multiparous transgenic female offspring from c-erbB-2-expressing lines develop a variety of focal hyperplastic and benign lesions that resemble lesions commonly found in human breasts. These lesions include lobular and ductal hyperplasia, fibroadenoma, cystic expansions, and papillary adenomas. More malignant lesions, including ductal carcinoma in situ and carcinoma, also develop stochastically at low frequency. The mammary glands of transgenic females invariably fail to involute fully after lactation. Similar phenotypes are observed in female MMTV-TGFα transgenic rats. In addition, multiparous TGFα-expressing female transgenics frequently develop severe pregnancy-dependent lactating hyperplasias as well as residual lobules of hyperplastic secretory epithelium and genuine lactating adenomas after weaning. These transgenic rat models confirm the conclusions reached in transgenic mice that overexpression of the c-erbB-2 and TGFα genes predisposes the mammary gland to stochastic tumor development. PMID:10393862

  10. Philosophical Reflection on Risks of Transgenic Technology

    Institute of Scientific and Technical Information of China (English)

    Xiaolu WANG

    2012-01-01

    Abstract [Objective] The aim was to analyze risks of transgenic technology. [Method] Discussions on risks of transgenic technologies were conducted from per- spective of philosophy. [Result] Mechanistic philosophy and reductionism are causes of reflection on risks of transgenic technology. Considering transgene is an artificial choice taking place of natural choice, it is inevitable for risks of transgenic technolo- gy to be found, in addition, social system constitutes the root for out-of-control of transgenic technology, hence, mechanism risk is the primary cause of transgenic risks. [Conclusion] It is inescapable for science view to be changed from arbitrary and lopsided to reflective and comprehensive and for technology view to be changed from exterminative and genesic to protective and symbiotic.

  11. Transgenic Epigenetics: Using Transgenic Organisms to Examine Epigenetic Phenomena

    Directory of Open Access Journals (Sweden)

    Lori A. McEachern

    2012-01-01

    Full Text Available Non-model organisms are generally more difficult and/or time consuming to work with than model organisms. In addition, epigenetic analysis of model organisms is facilitated by well-established protocols, and commercially-available reagents and kits that may not be available for, or previously tested on, non-model organisms. Given the evolutionary conservation and widespread nature of many epigenetic mechanisms, a powerful method to analyze epigenetic phenomena from non-model organisms would be to use transgenic model organisms containing an epigenetic region of interest from the non-model. Interestingly, while transgenic Drosophila and mice have provided significant insight into the molecular mechanisms and evolutionary conservation of the epigenetic processes that target epigenetic control regions in other model organisms, this method has so far been under-exploited for non-model organism epigenetic analysis. This paper details several experiments that have examined the epigenetic processes of genomic imprinting and paramutation, by transferring an epigenetic control region from one model organism to another. These cross-species experiments demonstrate that valuable insight into both the molecular mechanisms and evolutionary conservation of epigenetic processes may be obtained via transgenic experiments, which can then be used to guide further investigations and experiments in the species of interest.

  12. Novel transgenic rice-based vaccines.

    Science.gov (United States)

    Azegami, Tatsuhiko; Itoh, Hiroshi; Kiyono, Hiroshi; Yuki, Yoshikazu

    2015-04-01

    Oral vaccination can induce both systemic and mucosal antigen-specific immune responses. To control rampant mucosal infectious diseases, the development of new effective oral vaccines is needed. Plant-based vaccines are new candidates for oral vaccines, and have some advantages over the traditional vaccines in cost, safety, and scalability. Rice seeds are attractive for vaccine production because of their stability and resistance to digestion in the stomach. The efficacy of some rice-based vaccines for infectious, autoimmune, and other diseases has been already demonstrated in animal models. We reported the efficacy in mice, safety, and stability of a rice-based cholera toxin B subunit vaccine called MucoRice-CTB. To advance MucoRice-CTB for use in humans, we also examined its efficacy and safety in primates. The potential of transgenic rice production as a new mucosal vaccine delivery system is reviewed from the perspective of future development of effective oral vaccines.

  13. T cell immunity using transgenic B lymphocytes

    Science.gov (United States)

    Gerloni, Mara; Rizzi, Marta; Castiglioni, Paola; Zanetti, Maurizio

    2004-03-01

    Adaptive immunity exists in all vertebrates and plays a defense role against microbial pathogens and tumors. T cell responses begin when precursor T cells recognize antigen on specialized antigen-presenting cells and differentiate into effector cells. Currently, dendritic cells are considered the only cells capable of stimulating T lymphocytes. Here, we show that mature naïve B lymphocytes can be genetically programmed by using nonviral DNA and turned into powerful antigen-presenting cells with a dual capacity of synthesis and presentation of antigen to T cells in vivo. A single i.v. injection of transgenic lymphocytes activates T cell responses reproducibly and specifically even at very low cell doses (102). We also demonstrate that T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions. These findings disclose aspects in the regulation of adaptive immunity and indicate possibilities for vaccination against viruses and cancer in humans.

  14. T-Cell Mediated Immune Responses Induced in ret Transgenic Mouse Model of Malignant Melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Abschuetz, Oliver [Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim , Heidelberg 69120 (Germany); Osen, Wolfram [Division of Translational Immunology, German Cancer Center, Heidelberg 69120 (Germany); Frank, Kathrin [Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim , Heidelberg 69120 (Germany); Kato, Masashi [Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi 487-8501 (Japan); Schadendorf, Dirk [Department of Dermatology, University Hospital Essen, Essen 45122 (Germany); Umansky, Viktor, E-mail: v.umansky@dkfz.de [Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim , Heidelberg 69120 (Germany)

    2012-04-26

    Poor response of human malignant melanoma to currently available treatments requires a development of innovative therapeutic strategies. Their evaluation should be based on animal models that resemble human melanoma with respect to genetics, histopathology and clinical features. Here we used a transgenic mouse model of spontaneous skin melanoma, in which the ret transgene is expressed in melanocytes under the control of metallothionein-I promoter. After a short latency, around 25% mice develop macroscopic skin melanoma metastasizing to lymph nodes, bone marrow, lungs and brain, whereas other transgenic mice showed only metastatic lesions without visible skin tumors. We found that tumor lesions expressed melanoma associated antigens (MAA) tyrosinase, tyrosinase related protein (TRP)-1, TRP-2 and gp100, which could be applied as targets for the immunotherapy. Upon peptide vaccination, ret transgenic mice without macroscopic melanomas were able to generate T cell responses not only against a strong model antigen ovalbumin but also against typical MAA TRP-2. Although mice bearing macroscopic primary tumors could also display an antigen-specific T cell reactivity, it was significantly down-regulated as compared to tumor-free transgenic mice or non-transgenic littermates. We suggest that ret transgenic mice could be used as a pre-clinical model for the evaluation of novel strategies of melanoma immunotherapy.

  15. Transgene expression systems in the Triticeae cereals.

    Science.gov (United States)

    Hensel, Götz; Himmelbach, Axel; Chen, Wanxin; Douchkov, Dimitar K; Kumlehn, Jochen

    2011-01-01

    The control of transgene expression is vital both for the elucidation of gene function and for the engineering of transgenic crops. Given the dominance of the Triticeae cereals in the agricultural economy of the temperate world, the development of well-performing transgene expression systems of known functionality is of primary importance. Transgenes can be expressed either transiently or stably. Transient expression systems based on direct or virus-mediated gene transfer are particularly useful in situations where the need is to rapidly screen large numbers of genes. However, an unequivocal understanding of gene function generally requires that a transgene functions throughout the plant's life and is transmitted through the sexual cycle, since this alone allows its effect to be decoupled from the plant's response to the generally stressful gene transfer event. Temporal, spatial and quantitative control of a transgene's expression depends on its regulatory environment, which includes both its promoter and certain associated untranslated region sequences. While many transgenic approaches aim to manipulate plant phenotype via ectopic gene expression, a transgene sequence can be also configured to down-regulate the expression of its endogenous counterpart, a strategy which exploits the natural gene silencing machinery of plants. In this review, current technical opportunities for controlling transgene expression in the Triticeae species are described. Apart from protocols for transient and stable gene transfer, the choice of promoters and other untranslated regulatory elements, we also consider signal peptides, as they too govern the abundance and particularly the sub-cellular localization of transgene products.

  16. Gamma-irradiation enhances transgene expression in leukemic cells.

    Science.gov (United States)

    Vereecque, R; Saudemont, A; Wickham, T J; Gonzalez, R; Hetuin, D; Fenaux, P; Quesnel, B

    2003-02-01

    The majority of immunotherapy-based gene therapy protocols consist of ex vivo gene transfer in tumor cells. To prevent further in vivo growth, modified cells must be irradiated before reinjection into patients. The present study examines the effects of gamma-irradiation on transgene expression in transduced leukemic cells. Human and murine leukemic cells were transfected with retroviral vectors or plasmids carrying beta-galactosidase, GM-CSF or CD80 genes. Fresh leukemic cells from patients with acute myeloid leukemia (AML) were transfected with AdZ.F(pK7) adenoviral vector. gamma-irradiation at various lethal doses enhanced transgene expression in leukemic cell lines and fresh AML cells when the gene of interest was under CMV promoter but not when SV40 promoter was used. Oxidative stress also enhanced transgene expression and both irradiation and oxidative stress effects were inhibited by addition of N-acetyl-L-cysteine, a thiol anti-oxidant, indicating the involvement of reactive oxygen species. Transgene expression was also enhanced in vivo 48 and 120 h after subcutaneous injection of irradiated leukemic cells in syngeneic mice. These results show that a cell vaccine protocol using ex vivo gene transfer of transduced cells might be feasible in acute leukemia even if leukemic cells must be irradiated at lethal doses prior to reinjection to patients.

  17. Transgenic Plants as Sensors of Environmental Pollution Genotoxicity

    Science.gov (United States)

    Kovalchuk, Igor; Kovalchuk, Olga

    2008-01-01

    Rapid technological development is inevitably associated with many environmental problems which primarily include pollution of soil, water and air. In many cases, the presence of contamination is difficult to assess. It is even more difficult to evaluate its potential danger to the environment and humans. Despite the existence of several whole organism-based and cell-based models of sensing pollution and evaluation of toxicity and mutagenicity, there is no ideal system that allows one to make a quick and cheap assessment. In this respect, transgenic organisms that can be intentionally altered to be more sensitive to particular pollutants are especially promising. Transgenic plants represent an ideal system, since they can be grown at the site of pollution or potentially dangerous sites. Plants are ethically more acceptable and esthetically more appealing than animals as sensors of environmental pollution. In this review, we will discuss various transgenic plant-based models that have been successfully used for biomonitoring genotoxic pollutants. We will also discuss the benefits and potential drawbacks of these systems and describe some novel ideas for the future generation of efficient transgenic phytosensors.

  18. Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2.

    Science.gov (United States)

    Liu, Zhen; Li, Xiao; Zhang, Jun-Tao; Cai, Yi-Jun; Cheng, Tian-Lin; Cheng, Cheng; Wang, Yan; Zhang, Chen-Chen; Nie, Yan-Hong; Chen, Zhi-Fang; Bian, Wen-Jie; Zhang, Ling; Xiao, Jianqiu; Lu, Bin; Zhang, Yue-Fang; Zhang, Xiao-Di; Sang, Xiao; Wu, Jia-Jia; Xu, Xiu; Xiong, Zhi-Qi; Zhang, Feng; Yu, Xiang; Gong, Neng; Zhou, Wen-Hao; Sun, Qiang; Qiu, Zilong

    2016-02-04

    Methyl-CpG binding protein 2 (MeCP2) has crucial roles in transcriptional regulation and microRNA processing. Mutations in the MECP2 gene are found in 90% of patients with Rett syndrome, a severe developmental disorder with autistic phenotypes. Duplications of MECP2-containing genomic segments cause the MECP2 duplication syndrome, which shares core symptoms with autism spectrum disorders. Although Mecp2-null mice recapitulate most developmental and behavioural defects seen in patients with Rett syndrome, it has been difficult to identify autism-like behaviours in the mouse model of MeCP2 overexpression. Here we report that lentivirus-based transgenic cynomolgus monkeys (Macaca fascicularis) expressing human MeCP2 in the brain exhibit autism-like behaviours and show germline transmission of the transgene. Expression of the MECP2 transgene was confirmed by western blotting and immunostaining of brain tissues of transgenic monkeys. Genomic integration sites of the transgenes were characterized by a deep-sequencing-based method. As compared to wild-type monkeys, MECP2 transgenic monkeys exhibited a higher frequency of repetitive circular locomotion and increased stress responses, as measured by the threat-related anxiety and defensive test. The transgenic monkeys showed less interaction with wild-type monkeys within the same group, and also a reduced interaction time when paired with other transgenic monkeys in social interaction tests. The cognitive functions of the transgenic monkeys were largely normal in the Wisconsin general test apparatus, although some showed signs of stereotypic cognitive behaviours. Notably, we succeeded in generating five F1 offspring of MECP2 transgenic monkeys by intracytoplasmic sperm injection with sperm from one F0 transgenic monkey, showing germline transmission and Mendelian segregation of several MECP2 transgenes in the F1 progeny. Moreover, F1 transgenic monkeys also showed reduced social interactions when tested in pairs, as

  19. Integration mechanisms of transgenes and population fitness of GH transgenic fish

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    It has been more than 20 years since the first batch of transgenic fish was produced. Five stable germ-line transmitted growth hormone (GH) transgenic fish lines have been generated. This paper reviews the mechanisms of integration and gene targeting of the transgene, as well as the viability, reproduction and transgenic approaches for the reproductive containment of GH-transgenic fish. Further, we propose that it should be necessary to do the following studies, in particularly, of the breeding of transgenic fish: to assess the fitness of transgenic fish in an aqueous environment with a large space and a complex structure; and to develop a controllable on-off strategy of reproduction in transgenic fish.

  20. Tetracycline-inducible Expression Systems: New Strategies and Practices in the Transgenic Mouse Modeling

    Institute of Scientific and Technical Information of China (English)

    Yan SUN; Xigu CHEN; Dong XIAO

    2007-01-01

    To accurately analyze the function of transgene(s) of interest in transgenic mice, and to generate credible transgenic animal models for multifarious human diseases to precisely mimic human disease states, it is critical to tightly regulate gene expression in the animals in a conditional manner. The ability to turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedented flexibility in dissecting gene functions in health and disease. Pioneering studies in conditional transgene expression have brought about the development of a wide variety of controlled gene expression systems, which meet this criterion. Among them, the tetracycline-controlled expression systems (e.g. Tet-off system and Tet-on system) have been used extensively in vitro and in vivo. In recent years, some strategies derived from tetracycline-inducible system alone, as well as the combined use of Tet-based systems and Cre/lox P switching gene expression system, have been newly developed to allow more flexibility for exploring gene functions in health and disease, and produce credible transgenic animal models for various human diseases. In this review these newly developed strategies are discussed.

  1. Optimization of Biofuel Production From Transgenic Microalgae

    Science.gov (United States)

    2013-02-27

    AFRL-OSR-VA-TR-2013-0145 OPTIMIZATION OF BIOFUEL PRODUCTION FROM TRANSGENIC MICROALGAE Richard Sayre Donald Danforth...Technical 20080815 to 20120630 OPTIMIZATION OF BIOFUEL PRODUCTION FROM TRANSGENIC MICROALGAE FA9550-08-1-0451 Richard Sayre Donald Danforth Plant...BIOFUEL PRODUCTION FROM TRANSGENIC MICROALGAE Grant/Contract Number: FA9550-08-1-0451 Reporting Period: Final Report Abstract: We have compared the

  2. Expression Systems and Species Used for Transgenic Animal Bioreactors

    OpenAIRE

    Yanli Wang; Sihai Zhao; Liang Bai; Jianglin Fan; Enqi Liu

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm ...

  3. Expression Systems and Species Used for Transgenic Animal Bioreactors

    OpenAIRE

    Yanli Wang; Sihai Zhao; Liang Bai; Jianglin Fan; Enqi Liu

    2013-01-01

    Transgenic animal bioreactors can produce therapeutic proteins with high value for pharmaceutical use. In this paper, we compared different systems capable of producing therapeutic proteins (bacteria, mammalian cells, transgenic plants, and transgenic animals) and found that transgenic animals were potentially ideal bioreactors for the synthesis of pharmaceutical protein complexes. Compared with other transgenic animal expression systems (egg white, blood, urine, seminal plasma, and silkworm ...

  4. Potential molecular consequences of transgene integration: The R6/2 mouse example

    Science.gov (United States)

    Jacobsen, Jessie C.; Erdin, Serkan; Chiang, Colby; Hanscom, Carrie; Handley, Renee R.; Barker, Douglas D.; Stortchevoi, Alex; Blumenthal, Ian; Reid, Suzanne J.; Snell, Russell G.; MacDonald, Marcy E.; Morton, A. Jennifer; Ernst, Carl; Gusella, James F.; Talkowski, Michael E.

    2017-01-01

    Integration of exogenous DNA into a host genome represents an important route to generate animal and cellular models for exploration into human disease and therapeutic development. In most models, little is known concerning structural integrity of the transgene, precise site of integration, or its impact on the host genome. We previously used whole-genome and targeted sequencing approaches to reconstruct transgene structure and integration sites in models of Huntington’s disease, revealing complex structural rearrangements that can result from transgenesis. Here, we demonstrate in the R6/2 mouse, a widely used Huntington’s disease model, that integration of a rearranged transgene with coincident deletion of 5,444 bp of host genome within the gene Gm12695 has striking molecular consequences. Gm12695, the function of which is unknown, is normally expressed at negligible levels in mouse brain, but transgene integration has resulted in cortical expression of a partial fragment (exons 8–11) 3’ to the transgene integration site in R6/2. This transcript shows significant expression among the extensive network of differentially expressed genes associated with this model, including synaptic transmission, cell signalling and transcription. These data illustrate the value of sequence-level resolution of transgene insertions and transcription analysis to inform phenotypic characterization of transgenic models utilized in therapeutic research. PMID:28120936

  5. RNA helicase Belle/DDX3 regulates transgene expression in Drosophila.

    Science.gov (United States)

    Lo, Pang-Kuo; Huang, Yi-Chun; Poulton, John S; Leake, Nicholas; Palmer, William H; Vera, Daniel; Xie, Gengqiang; Klusza, Stephen; Deng, Wu-Min

    2016-04-01

    Belle (Bel), the Drosophila homolog of the yeast DEAD-box RNA helicase DED1 and human DDX3, has been shown to be required for oogenesis and female fertility. Here we report a novel role of Bel in regulating the expression of transgenes. Abrogation of Bel by mutations or RNAi induces silencing of a variety of P-element-derived transgenes. This silencing effect depends on downregulation of their RNA levels. Our genetic studies have revealed that the RNA helicase Spindle-E (Spn-E), a nuage RNA helicase that plays a crucial role in regulating RNA processing and PIWI-interacting RNA (piRNA) biogenesis in germline cells, is required for loss-of-bel-induced transgene silencing. Conversely, Bel abrogation alleviates the nuage-protein mislocalization phenotype in spn-E mutants, suggesting a competitive relationship between these two RNA helicases. Additionally, disruption of the chromatin remodeling factor Mod(mdg4) or the microRNA biogenesis enzyme Dicer-1 (Dcr-1) also alleviates the transgene-silencing phenotypes in bel mutants, suggesting the involvement of chromatin remodeling and microRNA biogenesis in loss-of-bel-induced transgene silencing. Finally we show that genetic inhibition of Bel function leads to de novo generation of piRNAs from the transgene region inserted in the genome, suggesting a potential piRNA-dependent mechanism that may mediate transgene silencing as Bel function is inhibited.

  6. Effect of β‑globin MAR characteristic elements on transgene expression.

    Science.gov (United States)

    Li, Qin; Dong, Weihua; Wang, Tianyun; Liu, Zhonghe; Wang, Fang; Wang, Xiaoyin; Zhao, Chunpeng; Zhang, Junhe; Wang, Li

    2013-06-01

    The aim of the present study was to investigate the effect of the characteristic elements of matrix attachment region (MAR) on transgene expression. Human β‑globin MAR was obtained by PCR amplification. A splicing MAR fragment containing all the characteristic elements of β‑globin MAR was artificially synthesized and then cloned into the eukaryotic expression vector. Following digestion and sequence identification, we transfected Chinese hamster ovary (CHO) cells with the two vectors, and then screened for the transformation of stable cells. The transgene expression level was analyzed by ELISA, and the copy numbers of the CAT gene were analyzed by real‑time fluorescent quantitative PCR. β‑globin MAR enhanced CAT reporter gene expression by 2.1489‑fold, whereas the β‑globin MAR characteristic elements did not enhance this expression. The real‑time fluorescent quantitative PCR analysis demonstrated that the relative copy numbers of the CAT gene of the β‑globin MAR expression vector were 1.2‑fold higher compared with those of the non‑MAR expression vector. MAR was able to improve the transgene expression level to a certain extent. The MAR characteristic elements did not improve the transgene expression alone. The transgenic expression levels were not linear with the transgene copy number; however, the enhancement of transgenic expression was relative to the increase in the gene copy number.

  7. Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

    Directory of Open Access Journals (Sweden)

    Shih Ping Yao

    2002-04-01

    Full Text Available Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C, is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57 of transgenic pigs (F0 generation. Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

  8. MAR elements and transposons for improved transgene integration and expression.

    Directory of Open Access Journals (Sweden)

    Déborah Ley

    Full Text Available Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

  9. MAR elements and transposons for improved transgene integration and expression.

    Science.gov (United States)

    Ley, Déborah; Harraghy, Niamh; Le Fourn, Valérie; Bire, Solenne; Girod, Pierre-Alain; Regamey, Alexandre; Rouleux-Bonnin, Florence; Bigot, Yves; Mermod, Nicolas

    2013-01-01

    Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

  10. Transgenic mosquitoes expressing a phospholipase A(2 gene have a fitness advantage when fed Plasmodium falciparum-infected blood.

    Directory of Open Access Journals (Sweden)

    Ryan C Smith

    Full Text Available BACKGROUND: Genetically modified mosquitoes have been proposed as an alternative strategy to reduce the heavy burden of malaria. In recent years, several proof-of-principle experiments have been performed that validate the idea that mosquitoes can be genetically modified to become refractory to malaria parasite development. RESULTS: We have created two transgenic lines of Anophelesstephensi, a natural vector of Plasmodium falciparum, which constitutively secrete a catalytically inactive phospholipase A2 (mPLA2 into the midgut lumen to interfere with Plasmodium ookinete invasion. Our experiments show that both transgenic lines expressing mPLA2 significantly impair the development of rodent malaria parasites, but only one line impairs the development of human malaria parasites. In addition, when fed on malaria-infected blood, mosquitoes from both transgenic lines are more fecund than non-transgenic mosquitoes. Consistent with these observations, cage experiments with mixed populations of transgenic and non-transgenic mosquitoes show that the percentage of transgenic mosquitoes increases when maintained on Plasmodium-infected blood. CONCLUSIONS: Our results suggest that the expression of an anti-Plasmodium effector gene gives transgenic mosquitoes a fitness advantage when fed malaria-infected blood. These findings have important implications for future applications of transgenic mosquito technology in malaria control.

  11. Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats.

    Science.gov (United States)

    Huang, S Z; Huang, Y; Chen, M J; Zeng, F Y; Ren, Z R; Zeng, Y T

    2001-09-01

    The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.

  12. Hydration kinetics of transgenic soybeans

    Directory of Open Access Journals (Sweden)

    Aline Francielle Fracasso

    2015-01-01

    Full Text Available The kinetic and experimental analyses of the hydration process of transgenic soybeans (BRS 225 RR are provided. The importance of the hydration process consists of the grain texture modifications which favor grinding and extraction of soybeans. The soaking isotherms were obtained for four different temperatures. Results showed that temperature affected transgenic soybeans´ hydration rate and time. Moisture content d.b. of the soybeans increased from 0.12 ± 0.01 kg kg-1 to 1.45 ± 0.19 kg kg-1 during 270 min. of process. Two models were used to fit the kinetic curves: an empirical model developed by Peleg (1988 and a phenomenological one, proposed by Omoto et al. (2009. The two models adequately represented the hydration kinetics. Peleg model was applied to the experimental data and the corresponding parameters were obtained and correlated to temperature. The model by Omoto et al. (2009 showed a better statistical fitting. Although Ks was affected by temperature (Ks = 0.38079 exp (-2289.3 T-1, the equilibrium concentration remained practically unchanged.

  13. Nematode neuropeptides as transgenic nematicides.

    Science.gov (United States)

    Warnock, Neil D; Wilson, Leonie; Patten, Cheryl; Fleming, Colin C; Maule, Aaron G; Dalzell, Johnathan J

    2017-02-01

    Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars.

  14. An inducible transgene reports activation of macrophages in live zebrafish larvae.

    Science.gov (United States)

    Sanderson, Leslie E; Chien, An-Tzu; Astin, Jonathan W; Crosier, Kathryn E; Crosier, Philip S; Hall, Christopher J

    2015-11-01

    Macrophages are the most functionally heterogenous cells of the hematopoietic system. Given many diseases are underpinned by inappropriate macrophage activation, macrophages have emerged as a therapeutic target to treat disease. A thorough understanding of what controls macrophage activation will likely reveal new pathways that can be manipulated for therapeutic benefit. Live imaging fluorescent macrophages within transgenic zebrafish larvae has provided a valuable window to investigate macrophage behavior in vivo. Here we describe the first transgenic zebrafish line that reports macrophage activation, as evidenced by induced expression of an immunoresponsive gene 1(irg1):EGFP transgene. When combined with existing reporter lines that constitutively mark macrophages, we reveal this unique transgenic line can be used to live image macrophage activation in response to the bacterial endotoxin lipopolysaccharide and xenografted human cancer cells. We anticipate the Tg(irg1:EGFP) line will provide a valuabl