TRANSCRIPTIONAL BEHAVIOR OF LCR ENHANCER ELEMENTS INTEGRATED AT THE SAME CHROMOSOMAL LOCUS BY RECOMBINASE-MEDIATED CASSETTE EXCHANGE

Efficient integration of transgenes at preselected chromosomal locatio ns was achieved in mammalian cells by recombinase-mediated-cassette-ex change (RMCE), a novel procedure that makes use of the CRE recombinase together with Lox sites bearing different spacer regions, We have app lied RMCE to the study of the human beta-globin gene Locus Control Reg ion by integrating at the same genetic locus in MEL cells, a LacZ gene driven by the human beta-globin promoter linked to HS2 and HS3 alone or in combination with HS4. Expression studies at the cell population level and in individual cells before and after induction of differenti ation with hemin or DMSO show that the presence of these enhancers is associated with variegated patterns of expression, We were able to sho w that the LCR fragments tested act by controlling both the probabilit y of expression and the rare of transcription of the linked beta-globi n promoter, Both of these factors were also dependent on the state of differentiation of the MELc and on the presence of a second transcript ion unit located in cis. The ability to manipulate by RMCE constructs integrated into chromosomes should help in the creation of complex, ra tionally designed, artificial genetic loci. (C) 1997 by The American S ociety of Hematology.