Cre/loxP-mediated excision of a neomycin resistance expression unit from an integrated retroviral vector increases long terminal repeat-driven transcription in human hematopoietic cells.
Recombinant retroviruses are currently the most attractive vehicles for gene transfer into hematopoietic cells. Retroviral vectors often contain an easily selectable marker gene in addition to the gene of interest. However, the presence and selection for expression of the selectable gene often result in a significant reduction of the expression of the gene of interest in the transduced cells. In order to circumvent this problem, we have developed a Cre/loxP recombination system for specific excision of the selectable expression unit from integrated retroviruses. A retroviral vector, containing both a neomycin resistance expression unit flanked by loxP sites and granulocyte-macrophage colony-stimulating factor cDNA, was used to transduce the human hematopoietic K-562 cell line. Four transduced cell clones were then superinfected with a retrovirus containing a Cre recombinase expression unit. Molecular analyses of 30 doubly transduced subclones showed a strict correlation between cre expression and loxP-flanked selectable cassette excision, thus implying that Cre recombinase activity is very efficient in a retroviral context. Moreover, the excision of the selectable cassette results in a significant increase of granulocyte-macrophage colony-stimulating factor transcription driven by the retroviral promoter.
Fernex
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C
Centre de Thérapie Génique, Institut Paoli-Calmettes, Marseille, France.
Dubreuil
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Mannoni
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P
Bagnis
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C
D000818:Animals; D002460:Cell Line; D004352:Drug Resistance, Microbial / Q000235:genetics*; D018014:Gene Transfer Techniques; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis; D006412:Hematopoietic Stem Cells; D006801:Humans; D019426:Integrases / Q000096:biosynthesis*; D019868:Kanamycin Kinase; D009115:Muridae; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae / Q000235:genetics*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014764:Viral Proteins; D016662:Virus Integration
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; C045073:Cre recombinase; D019426:Integrases