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Download DNAs for co-transfection into eukaryotic cells EUCLEX (In Vivo
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EUCLEX (In Vivo Eukaryotic Cloning and Expression System) Isolated pool of mRNAs AAAAA c-DNA synthesis with oligodT-attX2 -biotin primer followed by ligation of an IHFDNA cognate site - attX1 adapter attX1 scIHF cognate site c-DNA AAAA attX2 TTTTT biotin Enrichment of single-chain (sc)IHF-attX1 -adapter-carrying c-DNAs though IHF affinity purification Further selection through biotin moiety if desired, or size fractionation IHF site 1. c-DNA AAAA attX2 TTTTT attX1 DNAs for co-transfection into eukaryotic cells biotin λ Integrase Lentivirus Integrase 2. Expression vector for λ Int and lentiviral Integrase (regulated) 3. Insertion vector p RS LTR attY1 attY2 transcriptional stop RS ori bla IRES EGFP/NEO LTR RS = restriction site to generate linearized insertion vector from purified plasmids from E. coli IRES = Internal Ribosomal Entry Site co-transfection/transduction into eukaryotic cells Inside mammalian/plant cell c-DNA IHF DNA site AAAA attX2 TTTTT attX1 p attY1 LTR biotin attY2 ori bla IRES EGFP/NEO transcriptional stop LTR λ Inth/218 – mediated att site recombination episomal p attZ1 c-DNA attZ2 genomic p LTR ori bla IRES EGFP/NEO LTR LTR Lentivirus integrase – mediated genomic integration attZ1 c-DNA attZ2 ori bla IRES EGFP/NEO stable c-DNA expression LTR co-expression of GFP or other marker genes Desired selection protocol applied to stably transfected cells Identification of c-DNA and verification
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