Site-Directed Engineering of Defined Chromosomal Sites for Recombinant Protein and Virus Expression

Site-directed engineering of defined chromosomal sites.
Jun 30, 2009


In cell line development, the integration of transgenes into the chromosomal DNA of the host cell is a crucial step. Targeted introduction has many advantages over classical random integration procedures because of strong influences of the chromosomal surroundings on the expression of transgenes. Site-directed integration leads to predictable expression properties, avoids screening, is fast, provides high safety, and is thus the method of choice for the targeted integration of transgenes. Recombinase mediated cassette exchange (RMCE) using heterologous recombinases has been described as an efficient and reliable method to target (integrate and replace) transgenes by site-directed engineering of defined chromosomal sites for recombinant protein and virus expression. The article reports on the exploitation of defined chromosomal sites for the consistent production of highly biologically relevant proteins, namely the expression of viral vectors and antibodies.

Genetically modified cells are used for a wide variety of purposes, ranging from basic research to elucidate gene functions (e.g., in transgenic mouse models) to biotechnological applications like manufacturing antibodies. Efficient production of proteins and virus particles using mammalian cell lines usually relies on stable, albeit random integration of an expression construct into the cell's chromosomal DNA. After a construct is incorporated, its expression levels will be determined by neighboring genetic elements.1,2 To exploit a favorable genomic locus for expression of a given construct, intensive screening usually is required to identify integration sites that support high-level protein production. Frequently, screened clones are even engineered to maximize their expression potential. Gene amplification is a standard technology that aims to increase the gene copy number and thereby improve the production properties of a given cell clone. The genomic instability associated with gene amplification is a major challenge for the establishment of a stable and fully characterized producer cell line. Further, these current state-of-the art protocols are usually time consuming and importantly, must be repeated for each and any transgene to be expressed.

In comparison to the integration of numerous copies, a single copy of the gene of interest represents an advantageous scenario because of the possibility of full characterization of the integration site and the increased genomic stability of the producer cell line. The production of biopharmaceuticals from a single integration site is indeed a possibility, as already shown by the authors of this article and others. Single copy integration of transgenes in selected chromosomal sites provides the required expression strength and stability.3–6

The biotechnological exploitation of producer cell lines containing a single copy of the transgene of interest gained considerable importance following the introduction of RMCE. As specified below, this technology permits the rapid exchange of expression cassettes of choice in defined genomic surroundings. In this way, RMCE allows the manipulation of a single chromosomal locus that supports the desired expression level of the relevant protein and renders its repeated re-use feasible. In this way, this technology represents an excellent way to exploit defined genomic sites and is a breakthrough in cell engineering.