Download Chapter 16 - 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
•
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