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13 Cards in this Set
- Front
- Back
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Germ Line Therapy
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Transforming the genes in the egg or sperm
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Somatic Gene Therapy
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Transform the bad cells and introduce the wild type gene into a specific tissue within the whole organism
Example: A kid with cystic fibrosis Lets transform the cells in the lungs by inserting the wild type copy of CFTR but inserting the gene into a specific tissue to change somatic cells |
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Electroporation
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Take naked DNA and using an electrical pulse the DNA will enter the cell
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Microinjection
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A single cell is held by a vacuum and a needle punctures the membrane to insert DNA
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Biolistics
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Dehydrate DNA and then use a gun to shoot DNA into cells
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Viruses
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Transduction: Viral transfer
Transfection: Transform animal cells Viruses are infectious, it is very easy for the virus to inject DNA They are good vehicles to transform animals cells into foreign DNA |
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Function and Structure of AAV
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Attaches to the cell and the AAV genome integrates on a unique spot on chromosome 19
The virus does not replicate by itself; it requires co-infection which provides the proteins/molecules for it to reproduce Usually replicates with adeno-virus or herpes Structure Single stranded DNA with 7 genes Has inverted terminal repeats at both ends which play a role in replicating the genome and insertion at the specific locus at chromosome 19 |
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Restriction Endonuclease
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Provides the CFTR gene fragment
Cuts up the DNA |
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Mechanism of the PCR
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We want to break the hydrogen bonds in DNA by raising the temperature
The PCR will raise the temperature to 94 degrees for a minute or two and the strands will denature We now have specific single stranded DNA primers Lower the temperature to 45 – 65 degrees for 15 seconds so the primer can anneal to the strand with hydrogen bonding We then have DNA polymerase to extend the DNA in the 3’ direction at a temperature of 72 degrees |
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pBluescript
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Resistant to ampicillin, so if you mix it with a bacteria the bacteria will show resistance to ampicillin ... A bacteria with this plasmid would grow and without it would die
Origin of replication to replicate itself when its in the bacteria Lac Z to create B-galactosidease to metabolize sugars |
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Lac Z and the multi cloning site of pBluescript
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Lac Z spans the gene but is interupted by a multi-cloning site, which is a place where lots of enzymes can be used to clone things into that site
If you clone something in, you interrupt the lac Z gene, rendering it non functional |
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What is X gal and when does it turn blue?
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X gal is a sugar
It turns blue in the presence of b-galactosidease If the lac Z gene is interrupted and b galactosidease isnt made, X gal turns white |
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Bacteria on the same plate can turn blue or white. So what?
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White bacteria are harboring plasmids that have the cloned gene
Blue bacteria means that lacZ isn't interrupted so there is no piece of DNA in the cloning site of pBluescript |
