USING RMCE TO PRODUCE VIRUSES AND PROTEINS

We screened various cell lines including human 293 cells, mouse NIH3T3, Chinese hamster ovary (CHO), and baby hamster kidney (BHK) cells for integration sites that meet the requirements of stable and high expression. For this purpose, we implemented an advanced site-specific cassette replacement strategy that combines retroviral tagging and a positive selection trap with the Flp/FRT recombination.^9,10 To select exclusively cells that underwent the site-specific recombination, the tagging cassette contains a transcriptionally inactive selection marker that is only activated by correct site-specific integration. This strategy makes it possible to correctly identify recombined clones with an efficiency level of 90% or better.¹⁰ We established a set of cells with singly tagged chromosomal sites and used green fluorescent protein (GFP) to identify potent integration sites. Various tagging protocols were successfully applied.¹¹

Figure 3A. Predictable and homogeneous product expression after targeting a tagged locus A. The GFP-containing tagging cassette of a master cell line has been replaced by a targeting vector encoding lacZ. The GFP expression analysis (measured by FACS) and a lacZ staining protocol were performed before and after RMCE. Whereas the tagged cell population exhibits a high level GFP expression but is negative in lacZ staining, resulting subclones after RMCE homogeneously lost the GFP signal but gained the expression of lacZ.

Figure 3B. Predictable and homogeneous product expression after targeting a tagged locus B. Seven individual clones of the RMCE experiment described in A were analyzed by Southern blotting to confirm a correct cassette exchange. Therefore, chromosomal DNA of the clones was digested with DNA endonucleases, blotted, and probed against the marker gene. Only successfully targeted clones would show a 3 kb fragment and be clearly distinguished from the tagged parental clone The lacZ expression of these clones was quantified and compared to the parental cell line. As shown in the diagram, the expression behavior is highly homogenous among all analyzed isogenic subclones.

To translate the application of viral vectors into the clinic, it is mandatory to produce high titers of viral vector from a fully characterized producer cell line. For each therapeutic vector, a specific producer cell line must be generated. The titer critically depends on the site of chromosomal integration of the therapeutic vector. Thus, tremendous screening efforts must be undertaken to isolate clones that express the vectors in high concentrations and stably over time—a process that can take several months when conventional retrovirus packaging cell lines are used.¹⁶ We established a strategy based on RMCE to establish a new generation of producer cell lines. Upon selection for a chromosomal locus that supports retroviral vector insertion, a flexible retrovirus producer cell line was established that simplifies the isolation of highly productive producer clones.^3,4 This approach reduces laboratory time, increases safety, and in particular makes it possible to adjust viral vectors to the requirements of individual loci.¹⁷