19 Flashcards
I’ll
Oioin
Type one diabetes - Frequency- onset
1:5000
- childhood onset (mostly)
- autoimmune - body attacks B cells in pancreas thus can’t produce insulin
Both types if diabetes have
Genetic components to them
Type 2 diabetes
- 15% lifelong risk
- lifestyle/ environment
Diagram of type 1 diabetes
Progression of gene therapy knowledge in 1960
1922: insulin first isolated from pig and cattle
1950s: identified protein sequences/structure
- treatment for diabetes was purified insulin for pigs and cattle
Consequences (and why) of insulin purified from pancreas of cattle or pigs
- human amino acid sequence is similar but not identical
- immune responses range from local irritation to anaphylactic shock
- not always pure
(Cow insulin has 2 amino acid changes in alpha chain surrounding the disulphides bond, pig insulin has one extra amino acid at the C terminal of the peptide chain - the 3 amino acid changes in cow and one in pig at great enough to induce immune response if injected into human)
Therapeutic proteins - human sources being used - pathogen transmission
- issues around safety (pathogen transmission), yield and source of protein (tissue availability)
- factors collected for haemophilic patients ending up containing HIV or hepatitis - couldn’t screen for HIV- many hemphouliacs ended up with aids
- growth hormone collected from cadavas used for dwarfism (need many pituitary glands to treat a single patient increasing risk every time you take a sample) this transmitted prion proteins - mad cow disease - Jacob
Profession in gene therapy up to 2010
1922: insulin first isolated from pig and cattle
1950: insulin protein sequence ‘ structure identified
1973: recombinant DNA technology born
1977: insulin gene mapped too chromosome 11
1980: gene for insulin sequenced
1982: Eli Lilly produced the first recombinant insulin
What are recombinant DNA technologies
Joining bits of DNA together (sometimes form different species). These are inserted onto an organisms to produce (express) a useful protein.
(E.g : Aequorea Victoria - GFP - produced fluorescent glow in stress response - can be inserted into other animals
- beta barrel structure )
Plasmids are critical elements for…
… recombinant DNA technologies
What are plasmids
- (usually) circular pieces of double stranded DNA
- replicate independently of the hosts chromosomal DNA
Where are plasmids found
- common in bacteria
- also found in eukaryotes
What do plasmids provide?
-provide a benefit to hosts EG antibiotic resistance
Key components of recombinant DNA plasmids
- origin of replication
- antibiotic resistance
- promotor
(In recombinant DNA plasmids) Origin of replication allows…
Initiation of replication using host DNA polymerase
(In recombinant DNA plasmids) antibiotic resistance genes…
Allow selection of cells containing plasmid
(In recombinant DNA plasmids) what does the promotor do?
- drives expression of your favourite gene (e.g insulin or GFP) in cells with appropriate transcription factor machinery
GFP (or variant) =
Gene
Promotor allows
The expression of protein in a specific cell type or organism
Promotor differ - can have promorotrs theta are only expressed in bacteria, eukaryotes or specific cell types
SAME GFP (or variants) but expressed in different cell types
- need a different promotor to allow different expression
Where are restriction enzymes found and what do they do?
- naturally found in bacteria
- used as a defence system to degrade foreign DNA
- cut double stranded DNA at specific sequences
Most common restriction enzyme an what sequence does it cut at (FIRST STEP OF CUTTING AND PASTING DNA INTO PLASMIDS)
EcoRI enxyme
Cuts at GAATTC sequence between the G and the A on the 5’ end and the G and the A on the 3’ end
- ends up with an overhang
- can insert this into other DNA with complementary ends
The role of DNA ligase in cutting and pasting DNA into plasmids
- two DNAs have complementary base paring (DNA insert has a sticky end)
- DNA ligase catalyses the formation of the phosphodiester bone to repair nick in DNA backbone
Process of amplifying plasmids - transformation
- transformation is the transfer of plasmids into bacteria’s
- transformed bacteria selected by antibiotic resistance contained on plasmid
- expression of plasmid gene in bacteria (if bacterial promotor)
- amplification of bacteria and purification of DNA for down steam uses e.g. PCR, cloning, transfection into other cells or organisms
(Yap page)
- bacteria uses ORI to make many copies of the plasmid
What is transformation
- the transfer of plasmids into bacteria
Two primary methods of artificial transformation …
- chemical transformation (requires chemically components cells uses cations to increase the permeability of the bacterium cell wall increasing the chance of DNA acquisition - includes heat shock step)
- electroporaton ( involves the application of an electric field inducing holes in the cells perimeter allowing exogenous DNA to pass into cell)
Universal genetic code is essential for taking these plasmids and expressing genes in other species
Yes
The universal genetic code - what does it mean and what is its significance
- all organisms “read” the same codons as the same amino acids
- AUG = methionine
- UGA = stop codon
- significance = we can transform a human gene into bacteria and it will still make the same protein
Problems when cloning eukaryotic genes for expression in prokaryotes
- prokaryotic genes dont have in trials
- prokaryotes dont have the machinery to process eukaryotic introns
Instead: we take cDNA
- take mature DNA thats already been spliced, isolate that and reverse transcribe it using reverse transcriptase making cDNA from the mRNA (thats already spliced) and we use that for recombinant DNA for the plasmid
DO IT FOR ALL RECOMBINANT TECHNOLOGIES NOT JUST FOR BACTERIA as intervening sequences can be large
Recombinant DNA technologies combine DNA from…
Different species
Restriction enzymes and DNA ligase are used to
Manipulate DNA and bacteria and used to amplify DNA
A universal genetic code allows genes from one species…
To be expressed in another species
