Gene Cassettes for Directional Insertion at theSfiI Cleavage Site in the SV40 Replication Origin of Mammalian Expression Vectors

Asselbergs, Fred A.M.; Gandor, Christine R.; Widmer, Roland

doi:10.1006/abio.1996.0521

Cited by 3 publications

(3 citation statements)

References 3 publications

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“…The reporter cell lines were also supertransfected with pGRgpt, a plasmid with a gpt selection marker, which expresses the human glucocorticoid receptor (31). However, overexpression of the glucocorticoid receptor did not affect expression of NGF or the NGF reporter genes.…”

Section: Expression and Dexamethasone Regulation Of Reporter Genes In...mentioning

confidence: 99%

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector

Asselbergs

Grossenbacher

Ortmann

et al. 1998

Nucleic Acids Research

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Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.

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Section: Expression and Dexamethasone Regulation Of Reporter Genes In...mentioning

confidence: 99%

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector

Asselbergs

Grossenbacher

Ortmann

et al. 1998

Nucleic Acids Research

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“…The cDNA coding for human intestinal [Ala54]-FABP was obtained as previously described (8) and was subcloned in the NcoI/BamHI sites of pSF-SMC/sCD23 (18). Caco-2 cells were transfected using a commercially available polycationic transfection system (Superfect Transfection Reagent, Qiagen, Switzerland).…”

Section: Cell Transfectionmentioning

confidence: 99%

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells

Darimont

Gradoux

Persohn

et al. 2000

Journal of Lipid Research

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Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0.72% of cytosolic proteins in fibroblasts ( Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [ 14 C]palmitate showed a 34% reduction ( P Ͻ 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined. -Darimont, C., N. Gradoux, E. Persohn, F. Cumin, and A. De Pover. Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells.

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“…In addition, because dying cells cannot be separated from living cells in suspension, DNA and other components released from the dead cells cause SSF3 cells to clump together and otherwise inhibit growth. Isolation of stably transfected cell lines with SSF3KVN1 and DEXtreated SSFVN9 cells was compared to SSF3 cells in FCScontaining medium in a cotransfection experiment with luciferase reporter plasmid pGL3control (Promega) and pS-FmDHFR, which expresses a methotrexate-resistant dihydrofolate reductase (Asselbergs et al, 1996). No significant differences were observed in the number of dhfr + colonies obtained nor in the proportion of luciferase-expressing colonies.…”

Section: Applications Of Adherently Growing Serum-independent Cho Cellsmentioning

confidence: 99%

Conditionally adherent growth of serum-independent CHO cells for automated drug screening and biopharmaceutical production

Gandor¹,

Zang-Gandor²,

Flor

et al. 1999

Biotechnol. Bioeng.

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SSF3 is a CHO cell line adapted for growth in protein‐free medium. It grows in suspension unless serum‐derived attachment factors such as vitronectin are added to the medium. Serum‐independent cell lines, which adhere to the substrate after induction with dexamethasone or constitutively, were created by transfection with a human vitronectin gene under control of the mouse mammary tumor‐virus promoter. Substrate attachment and SSF3VN‐cell spreading could be prevented with an RGD peptide (arginine‐glycine‐aspartic acid) confirming that attachment is mediated by an intregrin receptor. Hormone‐inducible attachment could be blocked by glucocorticoid antagonist promegestone. All steps in the isolation of stable transfected SSF3VN cell lines could be done in a chemically defined medium avoiding the risk of introduction of serum‐derived infectious agents. SSF3VN cells could be grown in protein‐free medium in solid‐phase large‐scale bioreactors. Application in microplates as used in high‐throughput screening was demonstrated in an assay of Ca2+ release from internal stores induced by agonist‐binding to recombinant human metabotropic glutamate receptor hmGluR1b. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65:523–528, 1999.

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Gene Cassettes for Directional Insertion at theSfiI Cleavage Site in the SV40 Replication Origin of Mammalian Expression Vectors

Cited by 3 publications

References 3 publications

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells

Conditionally adherent growth of serum-independent CHO cells for automated drug screening and biopharmaceutical production

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