The isolation of CHO cells with a site conferring a high and reproducible transgene amplification rate.
J Biotechnol
; 164(2): 346-53, 2012 Dec 15.
Article
em En
| MEDLINE
| ID: mdl-23376841
ABSTRACT
Co-amplification of transgenes using the dihydrofolate reductase/methotrexate (DHFR/MTX) system is a widely used method for the isolation of Chinese hamster ovary (CHO) cell lines that secrete high levels of recombinant proteins. A bottleneck in this process is the stepwise selection for MTX resistant populations; which can be slow, tedious and erratic. We sought to speed up and regularize this process by isolating dhfr(-) CHO cell lines capable of integrating a transgene of interest into a defined chromosomal location that supports a high rate of gene amplification. We isolated 100 independent transfectants carrying a gene for human adenosine deaminase (ada) linked to a φC31 attP site and a portion of the dihydrofolate reductase (dhfr) gene. Measurement of the ada amplification rate in each transfectant using Luria-Delbruck fluctuation analysis revealed a wide clonal variation; sub-cloning showed these rates to be heritable. Site directed recombination was used to insert a transgene carrying a reporter gene for secreted embryonic alkaline phosphatase (SEAP) as well as the remainder of the dhfr gene into the attP site at this location in several of these clones. Subsequent selection for gene amplification of the reconstructed dhfr gene in a high ada amplification candidate clone (DG44-HA-4) yielded reproducible rates of seap gene amplification and concomitant increased levels of SEAP secretion. In contrast, random integrations of the dhfr gene into clone HA-4 did not yield these high levels of amplification. This cell line as well as this method of screening for high amplification rates may prove helpful for the reliable amplification of recombinant genes for therapeutically or diagnostically useful proteins.
Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes
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Transfecção
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Amplificação de Genes
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Células CHO
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Dosagem de Genes
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Transgenes
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article