lecture 19 Flashcards
1
Q
What is FokI?
A
- The enzyme FokI, naturally found in Flavobacterium okeanokoites, is a bacterial type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and a non-specific DNA cleavage domain at the C-terminal
- all restriction enzymes used in a lab come from bacteria - they are a way for bacteria to break down DNA that comes into it
- cuts the 5’ - 3’ strand 9 bases away from the recognition site (GGATG(N)) and the 3’ to 5’ strand 13 bases away (CCTAC(N)
- so after binding recognition sequence it doesn’t care what it is cutting 9/13 bases away, it just cuts
2
Q
What is CRISPR/Cas technology?
A
- based on an enzyme system
- in bacteria and archaea Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems provide immunity against foreign DNA based on RNA-guided DNA endonucleases
- the CRISPR/Cas9 system recognises and cleaves double stranded DNA complementary to a guide RNA (gRNA) sequence
- RNA-mediated genome editing
- you have this RNA molecule that finds a complementary site in the genome
- the cas9 binds to the RNA and that is binding to the sequence –> cas9 then able to create the cleavage
- most papers have used two plasmids: one that will make the RNA construct and one that will make the cas9 protein
- recently one construct that makes both of the things that you need
- two promoters: U6 (ribosomal RNA promoter) and CMV (a ubiquitously expressed viral promoter from the cytomegalovirus)
- the first drives the transcription of the RNA molecule
- other drives transcription of your cas9 molecule
- RNA molecule not converted into a protein while cas9 will
- this is injected into a fertilised egg
- as one construct makes life a lot easier
- cas protein contains two nucleases and both are required for cutting dsDNA
- spacer region of the RNA is where the researcher adds the nucleotide sequence of the gene of interest: 19-20bp cDNA from gene of interest to insert into spacer region of plasmid
- tracr RNA: same for all constructs, taken out of the bacterium and folds into this beautiful secondary structure – this is what the cas9 binds to
- together make the guide strand
CRISPR/Cas9 plasmid expression vector is tranfected into cells of interest
- the single vector produces guide RNA (gRNA) strand and Cas9 protein in the transfected cells
- once bound cuts the DNA upstream of the spacer region
- requirement of PAM sequence
- Cas9 detects genomic target by unwinding the DNA duplex, scanning for regions of complementarity between the genomic DNA and spacer sequence in guide RNA
- in order to cut the DNA, cas9 must detect spacer adjacent motif (PAM
- PAM is not part of the spacer nor any other part of the gRNA
- the tracr RNA has been engineered to mimic the hairpin in bacteria gRNA
- GG/CC
3
Q
What are issues with using CRISPR/Cas technology?
A
- the PAM sequence motif must be found adjacent to the spacer
it must contain a particular sequence (GN20 GG)
it must occur in the gene locus region you wish to make your spacer target - the CRISPR/Cas system can tolerate up to six mismatches in the spacer region leading to off-target cleavage
doesn’t have high fidelity
4
Q
What is a plasmid expression vector?
A
- bacteria have these naturally: self-replicating, circular pieces of DNA that can be up to 20 kb
- ones used in research are usually between 3 and 5 kb
- naturally occur in bacteria
- naturally occurring antibiotic resistance, resistance to other things
- have to keep the origin (ORI): this is what allows it to self replicate
- usually have some kind of antibiotic resistance genes
- need a promoter region: especially if you want it expressed in all cells something like CMV is useful (don’t know any cells it hasn’t worked in)
- cloning site that will have about 10 or 12 restriction enzymes that cut the plasmid and cut the gene of interest
- in between the polylinker/multiple cloning site you can put the gene of interest (GOI)
- create an intron: this allows the mRNA to be spliced, helps transport it into the cytoplasm so it can be translated
- polyA added to help it move out into the cytoplasm
- expression vectors = RNA + protein
5
Q
What is the transfection technique?
A
can use electroporation, liposomes
if you are using zygotes you will inject it – do not electroporate
- straight into pro-nucleus
6
Q
How can we tell which cells have taken up the plasmid?
A
- bacterial colonies grown on ampicillin-treated agar plates
- bacteria with ampicillin resistance gene survive and produce colonies
- those without the ampicillin resistance gene do not grow
- in mammalian cells use the G418 –> cells with neomycin will survive
- s genome
7
Q
What are problems encountered with plasmid expression vectors?
A
in yeast and mammalian cells:
- post-translational modifications possible
- saturation of translation and secretion processes
- low transfection efficiency
- toxicity to cells
- low fidelity
- sometimes difficult to isolate expressed protein
8
Q
What are applications of plasmid expression vectors?
A
- recombinant proteins made in E. coli, yeast or mammalian cells for human therapy
- insulin
- factor VIII, IX for haemophilia A
- human growth hormone
- erythropoietin for anaemia
- interferons
- interleukins
- granulocyte-macrophage colony-stimulating factor (to stimulate bone marrow after transplant)
- tissue plasminogen activator for dissolving blood clots
- etc - recombinant proteins for research e.g. to study function and cellular localisation
9
Q
Give a summary of transfection techniques
A
- transfection techniques (electroporation, liposomes, CaCl2) lead to very low transfection efficiencies: up to 50% of cells transfected, <1% stably transfected
- for some applications, higher transfection efficiencies are absolutely essential
10
Q
What are elements of a typical retrovirus?
A
- lipid bilayer
- envelope protein (env)
- capsid protein (gag)
- RNA
- reverse transcriptase enzyme
11
Q
How does the retroviral genome compare with the viral vector?
A
retroviral
- linear RNA molecule
- long terminal repeats on the ends (drives the expression of all the other proteins)
- packaging protein, gag, pol, env
vector
- take out gag, pol and env –> can’t replicate
- drives expression of whatever you put in there but won’t replicate
- only makes the RNA that is able to be converted into the protein
- integrates into the host genome
12
Q
What are elements of a typical adenovirus?
A
- adenoviruses are non-enveloped icosahedral particles
- dsDNA genome - linear
- coat - pentons and hexons
- fibers
- responsible for things like influenza
13
Q
How does the adenoviral genome compare with the vector?
A
adenoviral genome
- ITR - E1 - E2 - E3 - E4 - ITR
‘gutless’ adenoviral expression vector
- ITR-CMV-GOI-ITR
14
Q
Compare the two types of viral vectors
A
adenovirus vs retrovirus:
- linear ds DNA vs linear ss RNA
- Does not integrate into host cell genome – episomal vs integrates into host cell genome
- infects non-dividing and dividing cells vs infects dividing cells only
adenoviral vectors vs retroviral vectors
- up to 8kb GOI insert (same)
- transient expression only vs long term expression possible
problems adenovirus:
- immune response – inflammation and increased cytokines
- duration short
problems retrovirus:
- insertional mutagenesis and/or recombination with with wildtype virus
( but duration long)
15
Q
How do viruses work?
A
- viral genome enters host cell (if host cell is a bacteria, virus is a bacteriophage)
- viral genome is replicated and transcribed –> viral replication is a genetic process
- viral mRNAs are translated and proteins processed
- particles assemble isnide host, then burst or bud to exterior
- free particles in tissue or environment
repeat
16
Q
What are examples where gene therapy has been used in humans?
A
Jesse Gelsinger:
- adenovirus vector plus OTC transgene
- wasn’t able to break down amino acids in the liver due to a defect in a particular enzyme
- ended up dying
- 18 years old
- ornithine transcarbamylase (OTC)
- rare liver disorder – can’t break down NH4 –> basically can’t break down protein, always had a very strict diet
- single gene defect
- adenoviral vector
- recombinant vector injected directly into liver cells
- spread to bone marrow, spleen, nodes
- overstimulated the immune system – IL6
- chronic inflammation
- died within a few days
- immune system already has antibodies to influenza virus
Rhys Evans
- retrovirus vector plus X-SCID transgene
- survived the process
- 4 years old
- x-Severe combined immunodeficiency syndrome (x-SCID)
- gamma c receptor subunit of IL receptors - No T or NK cells
- “bubble boy” - no immune system
- single mutated gene (makes gene therapy a lot easier)
- retroviral vector
- removed lymphocytes at age 10 months
- transfected (infect) with recombinant vector
- returned to bone marrow
- effective immune system function
- several years later developed leukaemia (around 9)d
- cause: insertional mutagenesis
17
Q
What gene therapy clinical trials have occurred worldwide?
A
countries:
- USA - 1143
- Australia - 29
- China - 23
Genes transferred
- cytokine - 331
- suicide - 148
- receptors - 125
- replication inhibitor - 77
- tumour suppressor - 152
clinical phase phase I: 1076 phase II: 294 phase III: 63 phase IV: 2
vectors used adenovirus: 424 retrovirus: 365 herpes simplex: 58 adeno-associated: 86 lentivirus: 48
clinical phase: diseases
cancer: 960
cardiovascular: 137
infectious: 112
