Complex Designer Cells in Screens

Today, some 40% of current pharmaceuticals target G protein-coupled receptors (GPCRs), and this huge family of proteins remains central to ongoing and intense pharmaceutical research [48]. As yet, fewer than one hundred of the receptor genes out of 800-1000 members of the superfamily have been well characterized, so that additional promising targets should emerge in time.

Cell lines suitable for screens against drugs that target GPCRs require the receptors and - depending on the cell type and the subfamily of the receptor - either the corresponding or a promiscuous G protein alpha subunit [49]. Furthermore, for high-throughput acceptable machine readability an additional gene may be introduced, usually a reporter gene functionally linked to the investigated event. Convenient reporter systems include calcium-sensitive photoprotein (aequorin) or a P-arrestin2-linked GFP or artificially split reporter enzymes where subunits need to interact to produce a signal. To further complicate the situation, the cell line cannot be chosen based entirely on convenience because the endogenous expression of multiple receptors can interfere with the assay [50].

The generation of a cell clone with desired expression levels for a single foreign gene out of a pool of stably transfected cells requires a nontrivial effort. The task becomes formidable with the required co-expression of several foreign genes that need to assemble into a target of several subunits that triggers or inhibits reporter expression in response to drugs. Developments in promoter architecture and the prevention of de-activation of foreign sequences by cellular mechanisms, as well as improved methods for the introduction of expression cassettes by directed recombination, facilitate the generation of designer cells for very complex screens.

For historic reasons, viral promoters such as the SV40 early promoter, the Rous sarcoma virus (RSV) long terminal repeat (LTR) and, most frequently, the immediate early promoter from human cytomegalovirus (hCMV), are used to drive the expression of foreign genes. However, despite the 10- to 50-fold different promoter activity in transient assays (expression measured at 2-3 days post introduction of recombinant DNA), stable producer clones containing the strongest promoter (hCMV) have no clear advantage over clones derived with other viral promoters. Moreover, expression levels vary greatly between individual clones containing the same vector, and in many clones the expression declines with prolonged propagation. One explanation for this observation is that viral promoters integrated into the host genome preferentially become inactivated by DNA methylation or progressive deacetylation of histones H3 and H4 [51]. Both processes are linked: DNA methylation induces deacetylation of histones, making the region inaccessible to transcription factors, while extensive acetylation is able to prevent methylation at promoter sites [52]. In particular, the hCMV promoter is affected so strongly that only very few clones maintain expression at a medium or higher level. The search for stable and highly expressing clones after random integration of the vector, which makes cell line generation so tedious and time-consuming, simply identifies rare genomic sites that are protected from this effect. Accordingly, sequences from such sites such as the chicken HS4 insulator [53] or matrix attachment regions [54] can be inserted into the vector flanking the expression unit to achieve stable expression, even from the hCMV promoter.

Protective sequences are not necessarily separated elements, but in some cases represent an integral part of a promoter region. Such protected promoters have been found among ubiquitously expressed, housekeeping genes, for example the TATA box binding protein (TBP) promoter [55], elongation factor 1alpha promoter [56, 57], and the phosphoglycerate kinase or the elongation factor2 promoter (our data). Similar to viral promoters, they can be used across species and tissues. Specifically chosen combinations such as PGK, EF1 and EF2, allow the co-introduction of several genes from a single vector, thus avoiding competition between neighboring promoters [58]. While some of these promoters lag behind the hCMV promoter (this may actually advantageous when physiologic expression levels are important), others confer similar or higher activity (Fig. 7.2).

To reduce the effort for generation of multiple test lines even further, a system that does not require screening for individual producer clones would be of significant advantage. The influence of the genomic locus on expression level and stability makes homologous recombination and integration by site-specific recombinases attractive in cell line design. Typically, a reporter gene (e.g., P-galactosidase) linked to a site for a recombinase such as Flp or Cre is used to identify a preferable locus for integration. When this site is targeted by co-transfection of a shuttle vector containing the gene of interest and a recombinase site with a second vector expressing the recombinase, the reporter gene is separated from its promoter and becomes inactivated. Insertion of the gene in all surviving clones is assured by a promoter trap. The system and a number of cell lines with active loci labeled for insertion are commercially available from Invitrogen for Flp recombinase (Flp-In™).

The generation of new cell lines using this system is complicated by the integration of multiple vector copies at separate positions and, more frequently, as concatamers as a result of high cellular ligase activity Although copy numbers can be influenced by the amount ofDNA transfected and the transfection method used, single copy integrations are difficult to achieve. Moreover, not all loci expressing the reporter gene are easily accessible by the recombinase. The first problem can be addressed by vector insertion by retroviral gene transfer [59].

Fig. 7.2 The human EF2 promoter as an example of a cellular promoter that drives strong and stable expression in the majority of clones. pEF2 gfp was constructed replacing the hCMV promoter in pcDNA3 by a 3700-bp human elongation factor 2 promoter region and compared to the commonly used CMV promoter in the parental plasmid. Gfp expres sion was analyzed by FACS in pools of stably transfected Chinese hamster ovary (CHO) cells after low stringency G418 selection at 30 and 90 days after transfection. Clones were grouped according to expression levels. Whereas the rate of moderate- and high-producers drops substantially between 30 and 90 days for pcDNA3, it remains stable for pEF2gfp.

Fig. 7.2 The human EF2 promoter as an example of a cellular promoter that drives strong and stable expression in the majority of clones. pEF2 gfp was constructed replacing the hCMV promoter in pcDNA3 by a 3700-bp human elongation factor 2 promoter region and compared to the commonly used CMV promoter in the parental plasmid. Gfp expres sion was analyzed by FACS in pools of stably transfected Chinese hamster ovary (CHO) cells after low stringency G418 selection at 30 and 90 days after transfection. Clones were grouped according to expression levels. Whereas the rate of moderate- and high-producers drops substantially between 30 and 90 days for pcDNA3, it remains stable for pEF2gfp.

Alternatively, we are using a set of two consecutive recombination procedures to first identify clones that either already contain a single copy of the replacement cassette, or to reduce concatamers to a single copy (see Fig. 7.3). This procedure of consecutive recombination also confirms that the locus is accessible to targeting as the second step screens for expression levels. To favor gene exchange over excision, we use mutated frt sites that differ in the core sequence from the wildtype site [60]: these sites efficiently recombine with identical frt sites, but fail to interact with each other. We positioned a promoterless ATG-deficient neo gene outside of the replacement cassette that is activated by a minimal promoter, and an in-frame start codon was introduced with the recombination cassette.

Fig. 7.3 Minimizing the effort towards multiple recombinant cell lines with predefined features. Derivation of starter clones for efficient gene replacement by consecutive recombinations. As an example, BHK cells are shown. (a) Pools of clones expressing high gfp levels are generated using stringent double selection (FACS analysis, left). In a first recombination, gfp is replaced for alpha-1-antitrypsin (aat) inactivating selection markers MI and MII

Fig. 7.3 Minimizing the effort towards multiple recombinant cell lines with predefined features. Derivation of starter clones for efficient gene replacement by consecutive recombinations. As an example, BHK cells are shown. (a) Pools of clones expressing high gfp levels are generated using stringent double selection (FACS analysis, left). In a first recombination, gfp is replaced for alpha-1-antitrypsin (aat) inactivating selection markers MI and MII

and activating marker MIII. After recombination, most clones lack gfp expression, while in a fraction (containing multiple vector copies) gfp is still active (FACS analysis, center). Only clones that have undergone successful recombination and are devoid of gfp are ranked by aat expression (right). (b) The chosen clone is susceptible to replacement of aat by multiple target genes. Efficiency is ensured by reactivation of markers MI and MII.

In addition, the cassette contained a second selection marker which needs to be activated by the promoter residing at the integration locus, thus allowing for multiple exchange procedures.

For routine gene exchange the combined use of two selection markers assures a high efficiency of recombination. Variations in the expression level that still occur among clones originating from a single individual recipient cell can be attributed to perturbance of the architecture of a previously stable locus, caused by epigenomic phenomena. The phenomenon is less prominent when the cellular promoters are used instead of the CMV promoter. We have successfully incorporated the system into mouse 3T3, Chinese hamster ovary and baby hamster kidney cells, as well as designer cell lines of human and avian origin.

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