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Recombinant DNA

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.

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Recombinant DNA

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  1. 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

  2. Molecular cloning of a gene using a vector plasmid

  3. 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

  4. 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/

  5. Synthesis of cDNA using reverse transcriptase

  6. 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

  7. A plasmid for cloning & expression in E. coli Replication Cloning into plasmid Transcription Translation Selection Bensasson et al. 2004 Heredity 92:483

  8. Get the construct into the cells of the host organism From Purves et al. Life the Science of Biology, 6th ed.

  9. Gene therapy with recombinant retrovirus Campbell & Reece, Biology, 7th ed.

  10. 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

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