Gene Transfer

1. Gene Transfer By Natalia Restrepo

2. What is Gene Transfer? • Gene Transfer is the incorporation of new DNA into and organism's cells, usually by a vector such as a modified virus. (i.e. E.Coli) • It is used in gene therapy. • Its main purpose is to deliver a gene to a living cell. • In order for gene transfer to be successful, the modification has to be inheritable and the seed produced has to contain the modification. • Originally, vectors were used. A gene was inserted into a plasmid, which would enter the DNA and load the gene. The plasmid was used as a vehicle to insert a gene.

3. How do we get the DNA in? Make the cells competent (able to take up DNA) by: a) Chemical treatment - usually cold shock plus (E. coli) b) Electroporator - high voltage shock (yeast)

4. What happens to the DNA? Restriction - DNA is regarded as "foreign" and is degraded by restriction enzymes Recombination • incoming DNA must match host DNA • incoming genes replace original recipient genes a) if incoming DNA is linear it must recombine to survive b) if incoming DNA is circular has its own origin of replication therefore it can survive without recombination

5. DNA Ligase • This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments. • The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The backbone is still incomplete. • DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.

6. Restriction Enzymes These are enzymes that cut DNA at specific sites. They are properly called restriction endonucleases because they cut the bonds in the middle of the polynucleotide chain. Some restriction enzymes cut straight across both chains, forming blunt ends, but most enzymes make a staggered cut in the two strands, forming sticky ends.

7. Plasmids Plasmids are by far the most common kind of vector • They are short circular bits of DNA found naturally in bacterial cells. • A typical plasmid contains 3-5 genes and there are usually around 10 copies of a plasmid in a bacterial cell. • Plasmids are copied separately from the main bacterial DNA when the cell divides, so the plasmid genes are passed on to all daughter cells. • They are also used naturally for exchange of genes between bacterial cells so bacterial cells will readily take up a plasmid. • Because they are so small, they are easy to handle in a test tube, and foreign genes can quite easily be incorporated into them using restriction enzymes and DNA ligase.

8. This is a Plasmid

9. This is a Plasmid in the Bacterial Cell

10. Gene Transfer in Action .

11. DNA Ligase and complementary base pairing .