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An important consideration in the design of multigene delivery technology is the availability of suitable vectors to introduce multiple genes stably and stoichiometrically into living cells and co-express these genes efficiently. As a promising system for this purpose, we developed multi-cDNA expression constructs harboring two to three tandemly situated cDNAs in a single plasmid. The utility of this vector system is amplified by combining it with the psiC31 recombinase system which mediates site-specific integration of the genes into naturally occurring chromosomal sequences. By analyzing 55 psiC31-mediated integration events with five different constructs, each carrying one, two or three tandem cDNA expression cassettes, we identified 39 pseudo attP sites in the HeLaS3 chromosomes. All these sites share a common motif containing an inverted repeat and showing a similarity to the native psiC31 attP. The 36 integration events represented 27 different pseudo attP sites, suggesting the possibility of duplicate integration of the multigene expression plasmids into different genomic loci in a single cell. We demonstrated successive introduction of two different multi-cDNA expression plasmids into definite chromosomal pseudo attP sites, attaining integration of four cDNAs of known genomic constitution at precise genomic loci of a single HeLaS3 cell. The expression levels of these several transgenes were enhanced and made equally stable and robust by inserting the cHS4 insulator between genes.

Simultaneous Single Cell Stable Expression of 2-4 cDNAs in HeLaS3 Using .PHI.C31 Integrase System