BHK Cell Line

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Zhang Jingang - One of the best experts on this subject based on the ideXlab platform.

  • construction of coagulant factor vii gene expression vector and expression in BHK Cell Line
    Progress in Biotechnology, 2005
    Co-Authors: Zhang Jingang
    Abstract:

    In order to achieve the expression of recombinant human coagulant factor Ⅶ (rhF Ⅶ) in mammalian Cell Lines and to study its biological activity. The hF Ⅶ cDNA was amplified using PCR method, and the expression vector was constructed by subcloning method. After identification by DNA sequencing and obtaining a correct recombinant vector, the recombinant expression vector pIRES-F Ⅶ was transfected into BHK-21 Cell Line by means of lipofectin procedure. The resultant transfected Cells were selected by G418.The positive Cell clones were cultured and the culture supernatants were collected for identification by SDS-PAGE, Western blot and bioassays. The results showed that the bioactive rhF Ⅶ was expressed in BHK-21 Cell Line, and laid a foundation for further research.

Pierre Brugières - One of the best experts on this subject based on the ideXlab platform.

  • Neuroprotective gene therapy for Huntington's disease, using polymer-encapsulated Cells engineered to secrete human ciliary neurotrophic factor: results of a phase I study.
    Human Gene Therapy, 2004
    Co-Authors: Jocelyne Bloch, Anne-catherine Bachoud-lévi, Nicole Déglon, Jean-paul Nguyen, Catherine Bourdet, L. Winkel, Philippe Remy, Jean-pascal Lefaucheur, Stéphane Palfi, Pierre Brugières
    Abstract:

    Huntington's disease (HD) is a monogenic neurodegenerative disease that affects the efferent neurons of the striatum. The protracted evolution of the pathology over 15 to 20 years, after clinical onset in adulthood, underscores the potential of therapeutic tools that would aim at protecting striatal neurons. Proteins with neuroprotective effects in the adult brain have been identified, among them ciliary neurotrophic factor (CNTF), which protected striatal neurons in animal models of HD. Accordingly, we have carried out a phase I study evaluating the safety of intracerebral administration of this protein in subjects with HD, using a device formed by a semipermeable membrane encapsulating a BHK Cell Line engineered to synthesize CNTF. Six subjects with stage 1 or 2 HD had one capsule implanted into the right lateral ventricle; the capsule was retrieved and exchanged for a new one every 6 months, over a total period of 2 years. No sign of CNTF-induced toxicity was observed; however, depression occurred in three subjects after removal of the last capsule, which may have correlated with the lack of any future therapeutic option. All retrieved capsules were intact but contained variable numbers of surviving Cells, and CNTF release was low in 13 of 24 cases. Improvements in electrophysiological results were observed, and were correlated with capsules releasing the largest amount of CNTF. This phase I study shows the safety, feasibility, and tolerability of this gene therapy procedure. Heterogeneous Cell survival, however, stresses the need for improving the technique.

  • Neuroprotective Gene Therapy for Huntington’s Disease Using a Polymer Encapsulated BHK Cell Line Engineered to Secrete Human CNTF
    Human Gene Therapy, 2000
    Co-Authors: Anne-catherine Bachoud-lévi, Nicole Déglon, Jean-paul Nguyen, Jocelyne Bloch, Catherine Bourdet, L. Winkel, Philippe Remy, Moses Goddard, J.-p. Lefaucheur, Pierre Brugières
    Abstract:

    Huntington's disease (HD) is an autosomal dominant genetic disease with devastating clinical effects on cognitive, psychological, and motor functions. These clinical symptoms primarily relate to the progressive loss of medium-spiny GABA-ergic neurons of the striatum. There is no known treatment to date. Several neurotrophic factors have, however, demonstrated the capacity to protect striatal neurons in various experimental models of HD. This includes the ciliary neurotrophic factor (CNTF), the substance examined in this protocol. An ex vivo gene therapy approach based on encapsulated genetically modified BHK Cells will be used for the continuous and long-term intracerebral delivery of CNTF. A device, containing up to 106 human CNTF-producing BHK Cells surrounded by a semipermeable membrane, will be implanted into the right lateral ventricle of 6 patients. Capsules releasing 0.15-0.5 μg CNTF/day will be used. In this phase I study, the principal goal will be the evaluation of the safety and tolerability of...

  • neuroprotective gene therapy for huntington s disease using a polymer encapsulated BHK Cell Line engineered to secrete human cntf
    Human Gene Therapy, 2000
    Co-Authors: Annecatherine Bachoudlevi, Nicole Déglon, Jean-paul Nguyen, Jocelyne Bloch, Catherine Bourdet, L. Winkel, Philippe Remy, Moses Goddard, J.-p. Lefaucheur, Pierre Brugières
    Abstract:

    Huntington's disease (HD) is an autosomal dominant genetic disease with devastating clinical effects on cognitive, psychological, and motor functions. These clinical symptoms primarily relate to the progressive loss of medium-spiny GABA-ergic neurons of the striatum. There is no known treatment to date. Several neurotrophic factors have, however, demonstrated the capacity to protect striatal neurons in various experimental models of HD. This includes the ciliary neurotrophic factor (CNTF), the substance examined in this protocol. An ex vivo gene therapy approach based on encapsulated genetically modified BHK Cells will be used for the continuous and long-term intracerebral delivery of CNTF. A device, containing up to 106 human CNTF-producing BHK Cells surrounded by a semipermeable membrane, will be implanted into the right lateral ventricle of 6 patients. Capsules releasing 0.15-0.5 μg CNTF/day will be used. In this phase I study, the principal goal will be the evaluation of the safety and tolerability of...

Akira Nishizono - One of the best experts on this subject based on the ideXlab platform.

  • Molecular analysis of the mutational effects of Thai street rabies virus with increased virulence in mice after passages in the BHK Cell Line
    Archives of Virology, 2012
    Co-Authors: Phatthamon Virojanapirom, Pakamatz Khawplod, Artikaya Sawangvaree, Supaporn Wacharapluesadee, Thiravat Hemachudha, Kentaro Yamada, Kinjiro Morimoto, Akira Nishizono
    Abstract:

    QS-BHK-P7, street rabies virus, after passages in the BHK Cell Line, had an in vitro phenotype that distinguished it from its parental virus. Both viruses caused lethal infection in mice by central nervous system inoculation; however, only QS-BHK-P7 killed mice by the intramuscular route. We found four mutations, S23R and H424P in ectodomain of the glycoprotein (G), I1711 V in the polymerase genes, and another at the non-coding region between the phosphoprotein and matrix protein genes of QS-BHK-P7. None of the mutations in the G gene occurred in previously reported pathogenic determinants. The roles of mutations in particular non-coding regions remain to be elucidated.

Wei Shou Hu - One of the best experts on this subject based on the ideXlab platform.

  • exploring the transcriptome space of a recombinant BHK Cell Line through next generation sequencing
    Biotechnology and Bioengineering, 2014
    Co-Authors: Kathryn C Johnson, Andrew Yongky, Nandita Vishwanathan, Nitya M Jacob, Karthik P Jayapal, Chetan T Goudar, George Karypis, Wei Shou Hu
    Abstract:

    Baby Hamster Kidney (BHK) Cell Lines are used in the production of veterinary vaccines and recombinant proteins. To facilitate transcriptome analysis of BHK Cell Lines, we embarked on an effort to sequence, assemble, and annotate transcript sequences from a recombinant BHK Cell Line and Syrian hamster liver and brain. RNA-seq data were supplemented with 6,170 Sanger ESTs from parental and recombinant BHK Lines to generate 221,583 contigs. Annotation by homology to other species, primarily mouse, yielded more than 15,000 unique Ensembl mouse gene IDs with high coverage of KEGG canonical pathways. High coverage of enzymes and isoforms was seen for Cell metabolism and N-glycosylation pathways, areas of highest interest for biopharmaceutical production. With the high sequencing depth in RNA-seq data, we set out to identify single-nucleotide variants in the transcripts. A majority of the high-confidence variants detected in both hamster tissue libraries occurred at a frequency of 50%, indicating their origin as heterozygous germLine variants. In contrast, the Cell Line libraries' variants showed a wide range of occurrence frequency, indicating the presence of a heterogeneous population in cultured Cells. The extremely high coverage of transcripts of highly abundant genes in RNA-seq enabled us to identify low-frequency variants. Experimental verification through Sanger sequencing confirmed the presence of two variants in the cDNA of a highly expressed gene in the BHK Cell Line. Furthermore, we detected seven potential missense mutations in the genes of the growth signaling pathways that may have arisen during the Cell Line derivation process. The development and characterization of a BHK reference transcriptome will facilitate future efforts to understand, monitor, and manipulate BHK Cells. Our study on sequencing variants is crucial for improved understanding of the errors inherent in high-throughput sequencing and to increase the accuracy of variant calling in BHK or other systems. Biotechnol. Bioeng. 2014;111: 770–781. © 2013 Wiley Periodicals, Inc.

Seth D. Findley - One of the best experts on this subject based on the ideXlab platform.

  • ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration
    The EMBO Journal, 1996
    Co-Authors: Richard D. Palmiter, Toby B. Cole, Seth D. Findley
    Abstract:

    Abstract A cDNA encoding a second zinc transporter (ZnT-2) was isolated from a rat kidney cDNA expression library by complementation of a zinc-sensitive BHK Cell Line. The protein predicted from the open reading frame of ZnT-2 cDNA has 359 amino acids and initiates with a CTG codon. It resembles ZnT-1 (a plasma membrane protein that stimulates zinc efflux) in overall topology in that it has six membrane-spanning domains, a histidine-rich intraCellular loop and a long C-terminal tail; however, the overall amino acid identity is only 26%. Unlike ZnT-1, which is in the plasma membrane and lowers Cellular zinc by stimulating zinc efflux, ZnT-2 is localized on vesicles and allows the zinc-sensitive BHK Cells to accumulate zinc to levels that are much higher than non-transformed Cells can tolerate. Zinc was visualized within these vesicles with zinquin, a zinc-specific fluorescent probe. The intraCellular compartment that accumulates zinc is acidic as revealed by staining with acridine orange or LysoTracker. Prolonged exposure of Cells expressing ZnT-2 to zinc causes an accretion of intraCellular vesicles. We suggest that ZnT-2 protects these Cells from zinc toxicity by facilitating zinc transport into an endosomal/lysosomal compartment.

  • Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc.
    The EMBO Journal, 1995
    Co-Authors: Richard D. Palmiter, Seth D. Findley
    Abstract:

    A cDNA encoding a zinc transporter (ZnT-1) was isolated from a rat kidney cDNA expression library by complementation of a mutated, zinc-sensitive BHK Cell Line. This cDNA was used to isolate the homologous mouse ZnT-1 gene. The proteins predicted for these transporters contain six membrane-spanning domains, a large intraCellular loop and a C-terminal tail. ZnT-1 is homologous to zinc and cobalt resistance genes of yeast. Immunocytochemistry with an antibody to a myc epitope added to the C-terminus of ZnT-1 revealed localization to the plasma membrane. Transformation of normal Cells with a mutant ZnT-1 lacking the first membrane-spanning domain conferred zinc sensitivity on wild-type Cells, suggesting that ZnT-1 functions as a multimer. Deletion of the first two membrane-spanning domains resulted in a non-functional molecule, whereas deletion of the C-terminal tail produced a toxic phenotype. Mutant Cells have a slightly higher steady-state level of intraCellular zinc and high basal expression of a zinc-dependent reporter gene compared with normal Cells. Mutant Cells have a lower turnover of 65Zn compared with normal Cells or mutant Cells transformed with ZnT-1. We propose that ZnT-1 transports zinc out of Cells and that its absence accounts for the increased sensitivity of mutant Cells to zinc toxicity.