Cre-loxP mediated genoinic targeting to develop rapid and reproducible expression of recombinant proteins in mammalian cells.
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Expression levels of transgenes in mammalian cells show extreme variability between individual clones isolated from a single transfection. This is due to differing number of copies integrated into the genome and also from chromosomal "position effects". Therefore extensive screening is required to isolate a suitable cell line for high level expression of a recombinant protein. In this work, the Cre-loxP site-specific recombination system was investigated to eliminate such problems by directing the rapid targeting of any input DNA to a single pre-selected site in Chinese hamster ovary (CHO) cell line. Cre is a 38 kDa recombinase encoded by the bacteriophage P1 which mediates recombination between a pair of specific 34 bp target sequencesc alled loxP sites. The recombination reaction was first investigated in vitro to establish which kinetic parameters could be relevant for efficient gene targeting: a recombinant baculovirus was constructed with a hexa-histidine-cre fusion gene. The activity of Cre protein purified (by single step, hexa-histidine/nickel-bindinga, ffinity chromatography) from infected insect cells was verified by: (i) Cre-loxP interaction in gel retardation assays and (ii) Cre-mediated intramolecular excision between two loxP sites flanking a lacZ gene in a plasmid DNA. To investigate Cre-mediated targeting of an exogenous DNA to a chromosomal loxP site, three CHO cell lines were constructed, each carrying a loxP site between a ß-actin promoter and a secreted alkaline phosphatase (SAP) gene as a reporter. To demonstrate the targeting event, a promoterless lacZ/neor gene was cotransfected with either a cre plasmid or the purified Cre protein from the baculovirus/insect system. Proper targeting should activate expression of f 3- galactosidase from the chromosomal 0-actin promoter and give loss or reduction of SAP expression in G418 resistant transformants. Southern blot analysis showed targeted events mediated by both cre plasmid and recombinant Cre protein. This work should allow the development of a generic mammalian cell line by incorporating the Cre-loxP system for rapid and reproducible large scale production of recombinant proteins.
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