A new expression system for mammalian cells based on putative replicator sequences of the mouse and a truncated thymidine kinase gene.
Gene
; 73(2): 427-37, 1988 Dec 20.
Article
em En
| MEDLINE
| ID: mdl-3149608
ABSTRACT
We have constructed a new expression vector for mammalian cells. The vector contains a truncated tk gene for amplification under selective conditions, a sequence putatively supporting the replication of plasmid DNA in eukaryotic cells (murine autonomously replicating sequence) and an expression cassette for the cDNA to be studied. As a model cDNA we have used that of human tissue-type plasminogen activator (t-PA). Analysis of Hirt supernatants and chromosomal DNA from L cells, prepared six weeks after isolation of the clones indicated a 50- to 500-fold amplification of the expression construct in the cells. Concomitantly, the expression of t-PA was dramatically increased. Our data are consistent with episomal persistence of the expression construct, with a head-to-tail mode of integration into the mouse genome and with coexistence of both episomal plasmids and head-to-tail integrates. In tk-deficient cell lines other then L-cells, such as mouse mastocytoma or rat hepatoma cells, a strong selection against the persistence of the expression construct was noted. After long-term propagation of the L-cells under selective conditions the expression of the indicator gene continually decreases, but finally a constant plateau level of expression is established. Expression could be restored to the original level by blocking more efficiently the de novo synthesis of nucleosides.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Timidina Quinase
/
Transfecção
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Ativador de Plasminogênio Tecidual
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Genes
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Vetores Genéticos
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
1988
Tipo de documento:
Article