Targeted Integration

Cell lines stably expressing a protein product are typically generated by transfection followed by integration of the expression plasmid into the host genome via nonho-mologous recombination. Such integration is a random event and generates clones with a wide range of expression levels (Fig. 6), reflecting the gene copy number integrated and the transcriptional activity of the locus in which the copies were integrated (positional effect). The concept that high expression loci exist in the genome has lead to the development of several strategies for targeting gene integration specifically to these transcriptionally active loci, thus, introducing a controlled integration event.

The targeted integration approach is comprised of two steps. First, a transcrip-tionally active locus in the host genome is identified by transfecting a plasmid encoding a reporter gene and a selectable marker (employing vector design strategies described in Chapter 4) flanked by specific recombination target sites. Clones are selected under an appropriate selection pressure and a large screening is performed to identify a high expressing clone. In the second step, a plasmid carrying a product gene and a different selectable marker, flanked again by the recombination target sites, is transfected into the clone together with a second plasmid that transiently expresses a site-specific recombinase. This recombinase recognizes the specific sequence of the target sites and promotes a recombination event between them, termed homologous recombination. This event is highly specific and ensures integration of the product gene at the preexisting target site. All clones generated in this manner display an identical site of integration (isogenic clones) and should, therefore, express similar levels of the transgene. This approach eliminates the need for

Cho Cell Display
Figure 6 Fluorescent in situ hybridization of recombinant sequences integrated into the chromosomes of CHO cells. Photos are of two different cell lines resulting from random integration of the plasmid DNA during a single transfection event (See color insert p. 1.)

screening a large number of clones, and potentially minimizes/eliminates the need for amplification, thus shortening the time required to develop a highly productive cell line. In addition, once identified, the "marked" host cell line can be used for recombination and expression of any desired gene.

Several approaches have been investigated, using different recombinases, such as Cre recombinase from the bacteriophage PI (58), which recognizes Lox P sites, or Flp recombinase from Saccharomyces cerevisiae, which recognizes FRT sites (59). Both Lox P and FRT sites are 34 bp long and asymmetrical. Depending on the orientation of the recognition sites, the recombinase may catalyze excision/integration (recognition sequences are in the same orientation) or inversion (opposite orientation). For the application described here, the excision/integration reaction is utilized. Both recombinases have been shown to work efficiently in mammalian cells (58-61).

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