pIRESpuro3 contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV), which permits the translation of two open reading frames from one messenger RNA (1–3). After selection with puromycin, nearly all surviving colonies will stably express the gene of interest, thus decreasing the need to screen large numbers of colonies to find functional clones. To select for cells that express high levels of the gene of interest, the selective pressure for antibiotic resistance was increased by shifting the puromycin resistance gene downstream to a less optimal position for translation as directed by the IRES sequence (3). By decreasing the level of expression of the antibiotic resistance marker, the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the gene of interest, at high levels. The expression cassette of pIRESpuro3 contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS) that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (4), the ECMV IRES followed by the gene encoding puromycin resistance (puromycin-N-acetyltransferase; 5), and the polyadenylation signal from SV40. Ribosomes can enter the bicistronic mRNA at the 5′ end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker. ?
When using the pIRESpuro3 Vector, the antibiotic exerts selective pressure on the whole expression cassette; thus, a high dose of antibiotic will select for cells expressing a high level of the gene of interest. This selective pressure also ensures that the expression of the gene of interest will be stable