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Recombinant DNA • What is the basis of recombinant DNA technology? • How does one “clone” a gene? • How are genetically modified organisms (GMOs) created? • Illustration using CFTR gene Molecular cloning of a gene using a vector plasmid Methods for obtaining a gene of interest • Restriction endonuclease cleavage of genomic DNA – • Large fragments with introns, ready to ligate into vector Reverse transcription of mRNA to cDNA – • No introns, have to add restriction endonuclease cleavage sites PCR – – – Genomic DNA RT PCR of mRNA Can incorporate restriction endonuclease cleavage sites into primers Any DNA fragments cut with the same restriction enzyme can be spliced together ADP + Pi DNA Ligase ATP Jane Wang, Science Creative Quarterly, http://www.scq.ubc.ca/image-bank/ Synthesis of cDNA using reverse transcriptase Vectors for cloning and expression • DNA spliced into plasmid DNA can be replicated in cells • "Expression" vectors have regulatory DNA segments for cells to transcribe and translate inserted foreign DNA • Expression vectors are specialized for their host organism A plasmid for cloning & expression in E. coli Replication Cloning into plasmid Transcription Translation Selection Bensasson et al. 2004 Heredity 92:483 Get the construct into the cells of the host organism From Purves et al. Life the Science of Biology, 6th ed. Gene therapy with recombinant retrovirus Campbell & Reece, Biology, 7th ed. Issues with gene therapy • How to get the engineered gene into the right target cells at high efficiency • How to make the therapy last – stem cells? • How to avoid adverse consequences (cancer) from random integration of transgene • How avoid an immune response against the therapeutic gene or vector