Flp-Mediated Integration of Expression Cassettes into FRT-Tagged Chromosomal Loci in Mammalian Cells

The creation of recombinant mammalian cells with defined expression levels requires extensive screening. This is mainly due to the unpredictable site and copy number of the recombinant DNA in the hosts’ chromosomal DNA. The method presented here is based on the exchange of expression cassettes in a previously tagged site. Since the site of chromosomal integration remains the same in all targeted cells and since the copy number is reduced to one, the level of expression is highly predictable. The use of this method includes two steps. The first one, the tagging step, makes use of retroviral reporter constructs, which ensure single-copy integration of the tagging vector. Upon screening for cell clones with appropriate expression strength these cells will be targeted. The targeting construct harboring the gene of interest will precisely replace the tagging reporter cassette making use of the Flp recombinase.