Marcel

quires a separate construct for each combination of genes to be employed. Therefore, in order to accelerate the development of assays requiring simultaneous expression of multiple recombinant targets, we have used multiple independently replicating episomes (Fig. 3). We have found that the addition of a second, third, or fourth episomal construct does not affect expression, copy number, or steady-state RNA concentrations from episomes already contained within the cell (R.A.H., unpublished observations). By using multiple episomes, each containing a single recombinant gene of interest, one can transfect any combination of vectors at will in a procedure that we term ''combinatorial transfections.''

The choice of an appropriate cell line that contains characteristics desirable or even essential to a HTS format is an important part of the assay development process. Considerations such as the lineage from which the cell line was derived,

Figure 3 Schematic representation of episomal vectors described in this work. Vectors exist as a set of nearly identical constructs that differ only in selectable marker. Construct pE3hyg contains the hygromycin antibiotic resistance coding sequence, pE3pur confers puromycin resistance, etc. Resistance markers are abbreviated as follows: Zeo, zeocin; bla, blasticidin; oua, ouabain; and gpt, xanthine-guanine phosphoribosyl transferase. In cases where multiple episomes are used, pE3hyg contains ''gene 1,'' pE3pur contains ''gene 2,'' pE3zeo contains ''gene 3,'' etc.

Figure 3 Schematic representation of episomal vectors described in this work. Vectors exist as a set of nearly identical constructs that differ only in selectable marker. Construct pE3hyg contains the hygromycin antibiotic resistance coding sequence, pE3pur confers puromycin resistance, etc. Resistance markers are abbreviated as follows: Zeo, zeocin; bla, blasticidin; oua, ouabain; and gpt, xanthine-guanine phosphoribosyl transferase. In cases where multiple episomes are used, pE3hyg contains ''gene 1,'' pE3pur contains ''gene 2,'' pE3zeo contains ''gene 3,'' etc.

adherent properties, absence of interfering receptor subtypes or protein isoforms, or the presence of necessary ancillary protein subunit or signal transduction components are all fundamental. Various cell lines such as HEK293 (293EBNA), CV1, the somatic cell hybrid line D98/raji [19], the osteosarcoma line 143 [19], and numerous EBV+ or EBNA1+ lymphoid cells that already express EBNA1 can be readily obtained from Invitrogen or the American Type Cell Culture [16]. In the event that a cell type not already expressing EBNA1 is preferred, one need only simultaneously introduce into the cell line of choice two episomes, one encoding EBNA1 and a second encoding the desired recombinant gene. In this case, it is important that EBNA1 be transcribed from a strong promoter so that sufficient concentrations of EBNA1 will be reached rapidly enough to enable the cell to maintain both episomes before they are lost from the cell population or integrated into the host chromosome. Commercially available vectors generally do not contain a recognizable promoter driving expression of EBNA1, and we have observed instability of expression when using these constructs. The use of multiple episomes permits even further flexibility in the choice and use of cell lines for drug discovery. For instance, T-antigen can be introduced via one of the episomal complements in order to immortalize a desired cell type, or the coding sequence for the macrophage scavenger receptor can be introduced in order to confer a strongly adherent phenotype to cells in culture [20]. In all cases,

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