Recombinant DNA What is the basis of recombinant DNA technology? How does one “clone” a gene?...

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

Jane Wang, Science Creative Quarterly, http://www.scq.ubc.ca/image-bank/

DNALigase

ATP

ADP + Pi

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

ReplicationCloning into plasmidTranscriptionTranslationSelection

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