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 fo r gene transfer into hematopoietic cells, Retroviral vectors often con tain an easily selectable marker gene in addition to the gene of inter est, However, the presence and selection for expression of the selecta ble gene often result in a significant reduction of the expression of the gene of interest in the transduced cells, In order to circumvent t his problem, we have developed a Cre/loxP recombination system for spe cific excision of the selectable expression unit from integrated retro viruses, A retroviral vector, containing both a neomycin resistance ex pression unit Banked by loxP sites and granulocyte-macrophage colony-s timulating factor cDNA, was used to transduce the human hematopoietic K-562 cell line. Four transduced cell clones were then superinfected w ith a retrovirus containing a Cre recombinase expression unit, Molecul ar analyses of 30 doubly transduced subclones showed a strict correlat ion between cre expression and loxP-flanked selectable cassette excisi on, 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-st imulating factor transcription driven by the retroviral promoter.