These vectors contain four different internal ribosome entry site-selectable markers, allowing high-efficiency selection of transductants expressing the desired cDNA.
In the vector, the cytomegalovirus (CMV) promoter drives transcription of a bicistronic mRNA encoding both preproAM and the neomycin resistance cassette, separated by an internal ribosome entry site (IRES), to facilitate antibiotic selection of AM-expressing clones.
For synthesis of the lentivirus vector pgk:cryab, a full length clone of human cryab cDNA (NM_001885.1, Origene, Rockville, MD) was cloned into a modified lentiviral pgk vector [ 12] containing an internal ribosomal entry site (IRES) and eGFP as selection marker.
For generation of the targeting vectors, homology arms were PCR amplified from genomic DNA and cloned into a vector containing the internal ribosomal entry site β-galactosidase reporter gene, a neomycin selection marker and two thymidine kinase-negative selection markers.
In the construct, the reporter gene is followed by an internal ribosome entry site and a second marker that allows selection of stably transduced cells.
We also inserted the zeocin and blasticidin resistance genes after the internal ribosome entry site (IRES) so we could use these drugs for selection.
The lentiviral vector contains the GFP gene driven by the ubiquitin promoter followed by an IRES (internal ribosome entry site) sequence and an antibiotic resistance gene for the selection of infected cells.
Bicistronic expression of ZsGreen is facilitated by internal ribosomal entry site only when CD20 mutant gene was translated, enabling the efficient selection of transformed cells.
Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency.
To mitigate this, the GOI can be linked to the selection marker with the insertion of an internal ribosome entry site (IRES) [ 38].
