Rapid Establishment of G-Protein-Coupled Receptor–Expressing Cell Lines by Site-Specific Integration

Abstract

IntroductIon The conventional drug development process is expensive and time-consuming, and despite large investments in research and development activities, the success rate of bringing a new drug to the market is low. the most prominent target class is that of g-protein-coupled receptors (gpcrs), which cover about 60%-70% of recently developed drugs. 1 upon modulation by extracellular ligands, these cell surface receptors mediate the transduction of extracellular stimuli into intracellular signals. the hydrophobic core composed of seventransmembrane α-helices (7tm) is common to all gpcrs. 2 this large class comprises approximately 800 different receptors that are grouped in three families (classes a, B, and c).in general, the generation of cell lines stably expressing a transgene requires transduction of the host cell line and subsequent integration of the expression cassette into the host cell genome. this leads to a random distribution of integration sites.3 it is a fact that most genomic loci do not support high or consistent transgene expression. Both the level and stability of transgene expression are strongly dependent on the genomic surroundings of the integration sites. this "position effect" renders the generation of stable cell lines a cumbersome and often lengthy task involving extensive screening to identify those cell clones that display the desired properties. 4 the recombinase-mediated cassette exchange (rmce) strategy was introduced to overcome this limitation of cell line engineering (for review, see Wirth et al. 5 ). rmce can be best described as a two-step molecular "cut-and-paste" mechanism.6 in the first step, a reporter gene cassette is randomly integrated into a host cell line of interest. the expression of the reporter gene serves as a marker that permits screening for the desired expression characteristics (e.g., high and stable expression). once isolated and confirmed, those "tagged" clones function as master cell lines. a prerequisite for the use of such a master cell line is a single-copy integration of the tagging cassette. With the help of dna recombinases such as the cre, 7-9 the Flp recombinase, 10-12 or the phic31 integrase 13,14 the tagged genomic loci of the master cell lines can be recycled by the integration of any gene of interest. Because the recombinase-driven cassette exchange itself is a highly sitespecific event, all positive expression characteristics of the master cell line are transferred to the resulting subclonal producers. the aim of the study presented here is to employ the rmce strategy for the establishment of gpcr-expressing cell lines. the main application area of these cell lines is the drug 1 department of gene regulation and differentiation, hZi-helmholtz centre for infection research, Braunschweig, germany. 2 inscreeneX gmbh, Braunschweig, germany. 3 molecular devices (uK) ltd., Wokingham, uK. 4 european collections of cell cultures (ecacc), salisbury, Wiltshire, uK. the establishment of mammalian cell lines reliably expressing g-protein-coupled re...

Repeated integration of antibody genes into a pre-selected chromosomal locus of CHO cells using an accumulative site-specific gene integration system

“…Nehlsen et al (2009) also reported antibody gene integration into CHO cells using Flp-RMCE. Recently, site-specific integration into a target locus was applied for the evaluation of packaging cell lines producing retroviral vectors with high and predictable titer (Gama-Norton et al 2010) and for the establishment of recombinant G-proteincoupled receptor-expressing cell lines (Schucht et al 2011). In these studies, however, a transgene cassette was introduced into the genome only once.…”

3.4 Rational Approaches for Transgene Expression: Targeted Integration and Episomal Maintenance

“…Schucht and coworkers [84] used high expressing master cell lines to express G protein-coupled receptors (GPCRs). Wilke et al used RMCE in mutant CHO Lec3.2.8.1 cells to produce glycoproteins with the well-established glycosylation pattern in a homogenous form [93].…”

“…Once having tagged and expressing a hotspot with recombination target sites, integration of any transgene of interest can be achieved in a short time. Since screening is not required anymore, the cell line development is significantly speeded up [84,85].…”

Recombinant protein production from stable mammalian cell lines and pools

“…The main advantage of this system is that the recombinant cell lines have a single copy of the transgene at a genome site known to be transcriptionally active. Recently, RMCE was employed for the overexpression of several different antibodies [51] and GPCRs [52] from clonal CHO cell lines. RMCE can also be used stable cell pool generation following co-transfection and selection of the tagged host cells [50].…”