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  • 1
    Electronic Resource
    Electronic Resource
    Regulated autocrine growth of CHO cells (2000)
    Springer
    Cytotechnology 34 (2000), S. 39-46 
    Details
    ISSN: 1573-0778
    Keywords: autocrine growth ; CHO ; hGH production ; IGF-I ; lac ; metallothionein ; transferrin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The goal of this work was to engineer a CHO cell line capable ofautocrine growth in a fully defined protein-free medium. Thiswas accomplished by stable integration of the genes encodinginsulin-like growth factor I (IGF-I) and transferrin into thegenome of a CHO-K1 cell line. Thelac operator/repressorsystem was used to regulate the expression of the IGF-I gene with thelac operator sequence being placed upstream ofthe coding sequence for IGF-I. The expression of thelacrepressor protein was driven by a modified metallothioneinpromoter allowing repressor expression to be regulated by theculture medium. The cell line calledSuper CHOr (r for regulated) was able to grow in protein-free medium in an autocrine fashion with a doubling time of 20–24 hr,either attached to microcarriers or as aggregate suspensioncultures. Upon addition of metal to the culture medium, therepressor protein was produced and bound to the operatorsequences shutting down the expression of IGF-I and arrestingthe growth of the cells. Expression of the human growth hormone(hGH) gene and production of hGH was induced by the presence ofmetal ions. It was possible to release the cells from growtharrest in the presence of metal by the addition of isopropylβ-D-thiogalactopyranoside (IPTG), which prevented bindingof the repressor to its operator sequences. The ability to growCHO cells in fully defined protein-free medium and to be able toregulate their growth rate offers a number of advantages for theuse of these cells as hosts for the production of recombinantDNA derived proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008194529175
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  • 2
    Electronic Resource
    Electronic Resource
    Recombinant insulin-like growth factor-I (IGF-I) production in Super-CHO results in the expression of IGF-I receptor and IGF binding protein 3 (1998)
    Springer
    Cytotechnology 28 (1998), S. 91-100 
    Details
    ISSN: 1573-0778
    Keywords: autocrine growth ; CHO ; IGF-I ; IGFBP ; IGF-I receptor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Previously, we described the genetic construction Super- CHO, a cell line capable of autocrine growth under fully defined protein-free conditions. Super-CHO cells constitutively express insulin growth factor-I (IGF-I) and transferrin in sufficient amounts to support long-term, stable growth without the addition of exogenous growth factors, thus making it an ideal host for the production of recombinant biopharmaceuticals. although IGF-I has been successfully expressed in Chinese Hamster Ovary (CHO) cells, the long term effects of recombinant IGF-I expression have not been explored. In particular, the expression of the endogenous IGF-I receptor in response to IGF-I production has not been reported. We report here the transcriptional induction of the type I IGF receptor gene in Super-CHO. In addition, we examined the conditioned medium for the presence of IGF-I binding proteins. Ligand blot analysis reveals the presence of IGF binding proteins present in the medium conditioned by Super-CHO cells as well as CHO cells incubated in the presence of IGF-I. Furthermore, immunoaffinity reveals that Super-CHO expresses IGF binding protein-3 in response to IGF-I production. These results suggest the autocrine growth of Super-CHO involves a complex interaction of cell type specific factors which regulate its utility of IGF-I.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1008073513948
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  • 3
    Electronic Resource
    Electronic Resource
    Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium. Serum-free growth of IGF-I-producing CHO cells (1997)
    Springer
    Cytotechnology 24 (1997), S. 55-64 
    Details
    ISSN: 1573-0778
    Keywords: CHO ; IGF-I ; serum-free ; autocrine growth ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/106 cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007969502256
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  • 4
    Electronic Resource
    Electronic Resource
    Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO (1996)
    Springer
    Cytotechnology 21 (1996), S. 279-288 
    Details
    ISSN: 1573-0778
    Keywords: proinsulin processing ; CHO ; mutant human proinsulin ; cell culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365350
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  • 5
    Electronic Resource
    Electronic Resource
    Super-CHO—A cell line capable of autocrine growth under fully defined protein-free conditions (1996)
    Springer
    Cytotechnology 22 (1996), S. 139-146 
    Details
    ISSN: 1573-0778
    Keywords: CHO ; insulin ; IGF-1 ; transferrin ; autocrine growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Chinese Hamster Ovary (CHO) cells are widely used for the large scale production of recombinant biopharmaceuticals. Growth of the CHO-K1 cell line has been demonstrated in serum-free medium containing insulin, transferrin and selenium. In an attempt to get autocrine growth in protein-free medium, DNA coding for insulin and transferrin production was transfected into CHO-K1 cells. Transferrin was expressed well, with clones secreting approximately 1000 ng/106 cells/24h. Insulin was poorly expressed, with rates peaking at 5 ng/106 cells/24h. Characterisation of the secreted insulin indicated that the CHO cells were incompletely processing the insulin molecule. Site-directed mutagenesis was used to introduce a furin (prohormone converting enzyme) recognition sequence into the insulin molecule, allowing the production of active insulin. However, the levels were still too low to support autocrine growth. Further investigations revealed insulin degrading activity (presumably due to the presence of insulin degrading enzymes) in the cytoplasm of CHO cells. To overcome these problems insulin-like growth factor I (instead of insulin) was transfected into the cells. IGF-1 was completely processed and expressed at rates greater than 500 ng/106cells/24h. In this paper we report autonomous growth of the transfected CHO-K1 cell line expressing transferrin and IGF-1 in protein-free medium without the addition of exogenous growth factors. Growth rates and final cell densities of these cells were identical to that of the parent cell line CHO-K1 growing in insulin, transferrin, and selenium supplemented serum-free media.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353933
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