Molecular Genetics 25-30 Flashcards
(174 cards)
1
Q
Why do we use model organisms?
A
- Long history of use
- Easy to grow and maintain in the laboratory
- Easy to manipulate
- Short life cycle
- Small genome
- Sequenced genomes: ideas of what caused which phenotypes
- Many mutants
- Used to inform the biology of other species
- Extrapolate from simple organisms to more complex ones
- Exploited in biotechnology
2
Q
Features of E. coli
A
- Escherichia coli
- Main organism used to manipulate DNA
- Rod-shaped
- Lives in colon - pathogenic
- Rapid growth
- Stored in fridge
- Model PROKARYOTE
3
Q
What are examples of model prokaryotes?
A
E. coli
Bacillus subtilis
4
Q
Who transformed the first recombinant DNA into a prokaryote and when?
A
1972
Boyer and Cohen made the first recombinant DNA and transformed it into E. coli
5
Q
When was E. coli’s genome sequenced and how many genes does it have?
A
Sequenced in 1997
4000 genes
6
Q
Features of bacillus subtilis
A
- Rod-shaped
- Gram positive
- Found in soil and digestive system of ruminants
- Forms tough spores
- Commensal
- Used to study bacterial cell differentiation and chromosome replication
- Secretes proteins easily
- Used in biotechnology in large scale enzyme production
7
Q
What are examples of model unicellular eukaryotes (yeasts)?
A
Saccharomyces cerevisiae
Pichia pastoris
8
Q
Features of Saccharomyces cerevisiae
A
- First eukaryote sequenced (1996)
- 6000 genes
- Yeast doubles every 90 minutes
- Stored in fridge
- Has several plasmids
9
Q
What are 3 plasmids that Saccharomyces cerevisiae possesses?
A
- 2u plasmid (high copy) (YEp): plasmid used for cloning in yeast
- YAC (yeast artificial chromosome) plasmid (low copy) (YCp/CEN):
- Integrating plasmids (YIp)
10
Q
Features of pichia pastoris
A
- Used in protein production
- Similar growth conditions as S. cerevisiae
- Grows to a high density
- Sequenced genome (2009)
- Often used to produce large quantities of proteins for purification for analysis
- Vectors integrate into genome
11
Q
What does YEp stand for as the other name for yeast’s 2u plasmid?
A
Yeast episomal plasmid
12
Q
General properties of modified plasmids (basic cloning vector)
A
- Small (3-10kb)
- Gene for antibiotic resistance
- Easy to transfer from cell to cell
- Easy to isolate from host
- High copy number (100-150 copies of plasmid per cell)
- Easy to detect and select
- Multiple cloning site / polylinker where restriction enzymes can cut and inset DNA
- Able to screen recombinants
13
Q
Types of cloning vector
A
- Modified yeast plasmids
- Artificial chromosomes
- Lambda replacement vectors
- Cosmids and Bacmids
- Ti plasmid
14
Q
Properties of BACs, YACs and PACs (artificial chromosomes)
A
- Bacterial artificial chromosomes
- Yeast artificial chromosomes
- P1 bacteriophage artificial chromosomes
- Can insert really large DNA fragments (up to 2000 kb)
15
Q
Which out of PACs, BACs and YACs can take the largest fragment?
A
YACs
16
Q
What is an important feature of YACs (CENs)
A
They are shuttle vectors, meaning they can be shuttled between two types of organisms
17
Q
Example: using YAC as a shuttle vector between yeast and E.coli
A
- Two selectable markers (for yeast - auxotropic marker and E. coli - antibiotic resistance) required
- Need two open reading frames
- Plasmids will be circular in E. coli and linear in yeast
18
Q
Features of Yeast Integrating Plasmids (YIp)
A
- In S. cerevisiae
- Useful for stable integration of something into the yeast DNA
- Do not replicate independently
- Recombines into chromosome - to select the chromosome will have the non-functional version of a gene and the plasmid will have the intact version, which is how you select for if the chromosome has taken up the plasmid
- Transformation frequency low, but transformant is stable
19
Q
Features of lambda replacement vectors (aka bacteriophage vectors)
A
- Bacteriophage is pathogen of E. coli
- Has linear dsDNA with two cohesive ends (cod sequences) which attaches to E. coli cell and injects DNA in
- DNA is linear when packaged into protein head, with cohesive ends on each end. Once in the cell, these cos sequences are removed and DNA circularises, replicated and produces more phages which cause the cell membrane of E. coli to lyse
- Middle section removed and replaced with gene of interest
- Larger than typical cloning vector (37-52 kb)
- Difficult to manipulate
- Used in cDNA and genomic libraries
20
Q
Features of cosmid vectors
A
- Just highly modified lambda replacement vector
- Everything between cos sites is removed
- DNA up to 45kb can be inserted
- This is packaged up into lambda particles and then used to infect E. coli
21
Q
What is gateway cloning?
A
- Developed in the 1990s
- Uses lambda replacement vector to transfer between vectors
- Lambda can integrate into the genome
- Integrase cuts at two sites: attP (in the plasmid/phage DNA) and attB (in the bacterial DNA)
- DNA will then ligate and is inserted into the chromosome - the sites are then called attL and attR
- This reaction is REVERSIBLE due to Xis excisionase. This is what gateway cloning exploits
22
Q
Process of gateway cloning
A
- There are two vectors involved: an entry vector and a destination vector
- The entry vector is a basic cloning vector with a selectable marker (eg kanamycin), an origin of replication and a MCS. Gene of interest put into entry vector with BP clonase
- Instead of lacZ gene in the vectors, a toxic ccdB gene is used. E. coli will be killed if there is not an entry into the ccdB gene as the E. coli will express the toxic gene
- Gene of interest switched into destination vector using LR climate
23
Q
When is it better to use a shuttle vector for cloning rather than gateway cloning?
A
When you need to clone more than 4 fragments, as the gateway cloning method only allows you to clone up to 4 DNA fragments
24
Q
Process of cloning more than 4 fragments
A
Using a shuttle vector (usually 2u plasmid) which shuttles from yeast to E. coli
Vector has two selective markers and two origins of replication (one for E. coli one for yeast)
If you add 2u and yeast selectable markers you can get any plasmid to function in yeast
25
Outline vector construction in yeast
Use a 2u plasmid eg pRS426
1. Linearise fragment
2. Amplify fragments with complementary overhangs by PCR
3. Transform into yeast and yeast will assemble fragments
Can take 3 days - quite quick
26
Outline the transformation of yeast
1. Use LiAOc (lithium acetate) to make yeast cells competent
2. Add PEG (polyethylene glycol) and plasmid and PCR products
3. Allow to recover at 30 degrees
4. Heat shock at 42 degrees
5. Plate out for 3 days on selective medium
6. Recover plasmids from yeast via miniprep (need and enzyme to digest cell wall so plasmids can be removed)
27
What does competent mean?
When a cell becomes able to alter its genetics by taking up extracellular DNA from its environment and become transformed
28
Outline Gibson assembly
1. Design primers to add overhangs
2. Add master mix provided by kit and incubate
3. An exonuclease will remove the 5’ ends so fragments can anneal
4. Close and seal gaps with DNA polymerase and DNA ligase
29
When was yeast first transformed?
1978
30
How many fragments can the Gibson assembly clone?
Up to 15
31
Outline Golden Gate assembly
- Used type IIS restriction enzymes
- These cleave outside of their recognition sequence, creating four base flanking overhangs
- Add type IIS sites to primers and amplify your product
- The destination vector contains sites with complementary overhangs
32
How many fragments can golden gate assembly clone?
8-10 fragments
33
What type IIS restriction enzyme is often used in golden gate cloning?
Fok1
| Recognised the asymmetric sequence GGATG and makes a staggered cut further down the sequence
34
Outline the transformation of E. coli with the cloned vector
1. Make the E. coli competent
2. Incubate the E. coli to give it time to produce the proteins for antibiotic resistance or another marker
3. Plate the E. coli on a plate containing antibiotics
35
What are the two ways to make E. coli competent?
1. Permeabilise the cell wall using calcium chloride, keep on ice, heat shock
2. Electroshock cells - this also permeabilises the cell wall
36
How to extract and store the plasmid?
- Screen E. coli colonies (eg blue/white screening, colony PCR where you look for the presence of certain fragments)
- Do an E. coli miniprep to extract plasmid
- Verify plasmid by PCR, sequencing and digestion
- Store plasmid DNA at -20 degrees
- Store miniprep E. coli culture in glycerol and freeze in -80 degrees
- Repeat this process to produce more plasmid
37
What is the alternative to a miniprep if you need more plasmids than it can supply?
Do a midiprep
38
What are 3 model fungi?
Aspergillis niger, A. oryzae and A. nidulans
39
Features of model fungi
- Human pathogens (Aspergillosis = respiratory disease), mildew, used in fermentation
- Genomes sequenced in 2007, 37 mb
- Grow in 3-5 days
- Transformation is via electroporation (electric shock to take up DNA), PEG-mediated or Agrobacterium
40
How is citric acid produced today?
With Aspergillus niger
41
What was the old method of producing citric acid?
Calcium citrate produced and then converted to citric acid chemically
42
What microbe produces monosodium glutamate (MSG)?
Corynebacterium glutamicum
43
What is Corynebacterium glutamicum also used to produce besides MSG
Lysine - an essential amino acid only found in small quantities in cereal crops. It is added to animal feed. Mutants were found that accumulated lysine under limiting conditions, but the enzyme that synthesisers lysine is inhibited under high lysine conditions. New mutants were found where this inhibition was defective.
Also used in production of methionine
44
Why is the chemical synthesis of vitamin C not preferred?
Chemical synthesis produces D- and L- forms of amino acids
| Humans can only taste L-forms and only L-forms can be incorporated into proteins, so half of the product is wasted
45
Why is the biological synthesis of vitamin C preferred?
Microorganisms only produce L- forms
46
What does the bacterium Gluconobacter oxydans do?
Convert sorbitol to sorbose - an intermediate step in the process of producing vitamin C
47
What was the process devised in the 1930s of producing vitamin C
Combination of chemical and biological methods to produce vitamin C
1. Nickel catalyst produces D-sorbitol
2. G. oxydans converts sorbitol to sorbose
3. Acid converts sorbose to 2-KLG
4. 2-KLG concerted with an acid to vitamin C
48
What is the starting mixture used to produce vitamin C?
Glucose
49
Outline the modern process of producing vitamin C
- The bacterium Erwinia sp. can convert glucose to 2.5 DKG
- Corynebacterium sp. can convert 2.5 DKG to 2-KLG
- Problem: different growth conditions needed
- Solution: transfer the gene from Corynebacterium sp. into Erwinia sp. so only one growth condition needed
- Acid treatment then converts 2-KLG to vitamin C
50
Advantages of recombinant protein production using E. coli
- Simple to grow
- Can grow in large bioreactors
- Cheap
- No ethical issues
- Safe
- High yields
51
Disadvantages of recombinant protein production in E. coli
- Codon bias
- High production can be toxic: kills E. coli culture
- Poor folding which leads to inclusion bodies being produced
- Poor secretion
- Post-translation differences
52
Why is codon bias a disadvantage of using E. coli to produce recombinant proteins? What is the solution?
- Redundancy in genetic code
- In E. coli, AAA is used to code for lysine most of the time
- If the sequence contains AAG instead, low amounts of the protein will be produced
- This can be overcome by site-directed mutagenesis of the DNA sequence, or synthesising the DNA, or feeding the culture with a supply of the missing tRNAs, or transforming the relevant genes into strains of E. coli
- Codon optimisation improves yield by about 70% when expressing human proteins in E. coli
53
Why is poor secretion a disadvantage of using E. coli to produce recombinant proteins? What is the solution?
- E. coli secretes few proteins
- Secretion into the culture medium facilitates protein recovery
- This is done by fusing the protein to a protein that is usually secreted or adding additional secretion sequences
- You could also modify E. coli to have a better extracellular secretory system
54
Why are post-translation modification issues a problem when using E. coli to produce recombinant proteins? What is the solution?
- Glycosylation is required for the proper function of many proteins
- Adding sugar residues to protein
- Prokaryotes add glycosyl groups to side chains, rather than N-terminal groups which is where eukaryotes add them
- The prokaryotes are therefore adding the glycosyl groups to the wrong place for human proteins
- The solution is taking glycosylation genes from Campylobacter and adding them to E. coli so your proteins will be glycosylated correctly
55
Insulin production in E. coli (old method)
1. Clone and express genes for insulin strands A, B and C in E. coli
2. This left preproinsulin with no post-translational modification
3. Put gene for A and B chains on two separate plasmids and put these into separate E. coli. Fused the proteins to B-galactosidase to ensure chains were secreted from the cell
4. Once proteins were collected they were chemically treated with cyanogen bromide to cleave off B-galactosidase
5. Oxidisation step required to form disulphide bonds and produce active insulin
56
Natural insulin production
1. Insulin strands B, C and A translated to give linear peptide
2. C strand cleaned off
3. Disulphide bonds form between strand A and B, leaving the complete insulin molecule
57
Advantages of using yeast to produce insulin
- Simple to grow
- Can grow in bioreactors
- Cheap
- No ethical issues
- Safe
- Glycosylates secreted proteins
- Improved folding - disulphide bonds form
58
Modification of porcine insulin for human use
- Allergenic reactions to porcine/bovine insulin
- Use enzymes to modify protein
- Trypsin cleaves next to arginine and lysine
- In porcine insulin, the terminal alanine is preceded by lysine
- Trypsin replaces the cleaved alanine with threonine - makes it into human form of insulin
59
Modern way to product insulin in E. coli
B, C and A strands synthesised, cloned in one plasmid and produced in one culture to produce insulin
Proinsulin digested with tryposin to remove C peptide and yield insulin
60
Disadvantages of using yeast to produce insulin
- Low yield
- Poor secretion
- Introns not spliced out as yeast is prokaryote
- Excessive glycosylation
61
What is yeast used to produce (medically)?
- Insulin
- Growth factors
- Blood clotting factors
- Viral proteins used in vaccines
62
How to solve the problem of insulin being prone to clotting?
- Clumping covered the receptor bonding site so reduces efficiency
- Not a problem in vivo as is secreted before it can clump
- Problem when injected
- Replace proline with asp
- These amino acids have negative charges so repel each other, preventing clumping
63
Features of Arabidopsis thaliana
- Thale cress
- Most widely used model in plant genetics
- Similar genetic responses to stress and disease as food crops
- Genome sequenced in 2000, 25,000 genes
- 5 chromosomes
64
What makes A. thaliana such a useful model plant?
6-8-week life cycle
Easily transformed
Many seeds produced
65
What are the two species of the second model plant genus?
Nicotiana benthamiana
| Nicotiana tabacum
66
What features make Nicotiana sp. such useful model organisms?
- Often used in virology and tissue culture
- Easy to cross
- Thousands is seeds per cross
- 3-4 month life cycle
67
How to make a transgenic plant:
1. As plant cells are totipotent, you can regenerate a whole plant from one cell
2. These cells are grown in tissue culture
3. There are various methods of transformation
68
What are the 3 methods of tissue culture to clonally propagate plant cells?
1. Plant leaf discs: take section of leaf and growing it in a petri dish. Hormones required to stimulate growth of shoots and roots
2. Callus capture: a mass of undifferentiated cells from an immature embryo or meristem, also treated with hormones to induce growth
3. Suspension culture: important for producing recombinant proteins. Protoplasts (call wall removed) or immature pollen placed in suspension and grown for several years in culture
69
Features of Agrobacterium tumefaciens
Plant pathogen
Gram negative bacteria
Soil dwelling
Causes crown gall
Attracted by phenolics and enters through plant wounds
Induced plant to produce excess auxin and cytokinin
Galls contain opines synthesised by genes on the Ti plasmid
70
Features of Ti plasmid of Agrobacterium tumefaciens
Tumour-inducing plasmid
200 kb
Cytokinin and auxin oncogenes and opine gene transferred into plant as T-DNA
the rest of the plasmid contains genes responsible for moving the T-DNA into the plant cell (virulence genes)
71
How does Agrobacterium infect a plant cell?
1. Wounded plant cell produces signal molecule
2. Receptors recognise signal molecules
3. Attaches to plant cell
4. Activated VIR proteins process T-DNA
5. T-DNA complex forms and transfers into plant cell
6. T-DNA moves into nucleus
7. Expression of bacterial proteins
72
Describe the genetics behind the Agrobacterium infection of a plant
1. VirA receptor detects acetosyringone and auto-phosphorylates
2. VirA phosphorylates transcription factor VirG, which initiates transcription of Vir genes B-E
3. VirD1 and VirD2 are DNA nickases which cut and release single-stranded T-DNA
4. VirD2 then attaches to the 5’ end of the T-DNA
5. VirE2 coats the T-DNA
6. T-DNA exits the bacterium and enters the plant. VirE2 and VirD2 interact with plant proteins
7. VirE2 and VirD2 have NLS (nuclear localisation signals) and the T-DNA complex moved to the nucleus
73
What is used instead of a Ti plasmid when using vector to transform Agrobacterium in molecular genetics?
A binary vector e.g. pCAMBIA
Has selectable marker, E. coli ORI and Agrobacterium ORI
We can insert a plant selectable marker and genes of interest between the left and right T-DNA borders
74
Process of producing a transgenic plant with a binary vector
1. Make plasmid with genes of interest
2. Transform plasmid into E. coli
3. Plasmid miniprep from E. coli
4. Transform plasmid into Agrobacterium
5. Co-cultivate Agrobacterium and plant tissue
6. Use antibiotics to kill off Agrobacterium and select transformed plant cells
7. Transgenic plant produced
75
How to use Agrobacterium to transform leaf discs
1. Discs removed from plant
2. Incubate leaf discs with Agrobacterium
3. Select for transformants and kill Agrobacterium
4. Transfer to shoot-inducing medium
5. Transfer to root-inducing medium
6. Transformed plant
76
What is the Agrobacterium floral dip method?
- Can use with Arabidopsis
- Dip inflorescences into an Agrobacterium culture
- Inflorescent cells are transformed prior to seeds being produced
- Seeds can then be screened for transformants by growing on selective medium
77
What is the gene gun / particle bombardment method of plant transformation?
- Gold particles are coating in the DNA construct to be introduced, then they are fired at target cell
- Gold is an inert substance so doesn’t affect the cell
- Some gold particles will reach the cell and the DNA can unwind and migrate to the nucleus
- Cells are then regenerated into whole plants
78
How to verify a plant has been transformed?
- Selectable marker, but this is not enough proof on its own
- PCR to see if the right-sized band is present
- Sequencing the PCR product to check the sequence is correct
- Reporter gene checks gene is being expressed
79
What are the 4 main GM crops produced globally?
soybean
maize
cotton
oilseed rape
80
What are the two main GM crop traits?
- Herbicide tolerance
| - Insect resistance
81
What is glyphosate and how can you transform a plant to become resistant to it?
- Glyphosate is a herbicide which blocks the synthesis of some amino acids by binding to an enzyme called EPSPS
- Introduce a mutant EPSPS from Agrobacterium tumefaciens (aroA) so glyphosate cannot bind
- Plant is now resistant to glyphosate so it will only kill the weeds around it
82
What is the Bt toxin and how can it be used to prevent plants being eaten by insects?
- cry genes from soil bacterium Bacillus thuringiensis code for a toxic crystal protein, which solubilises when ingested by insects
- It binds to epithelial cells within the digestive tract and creates holes
- Insects die within a few days
83
Other GM traits
- Virus resistance
| - Quality traits
84
Examples of quality traits that are genetically modified into plants
- Drought resistance
- Lysine biosynthesis
- Photosynthetic genes
- Golden rice
85
What are second generation GM crops
Crop that have been genetically modified to be of a higher quality - less important than insect resistance and herbicide tolerance
86
What are third generation GM crops?
Crops used for manufacturing pharmaceuticals - biopharming
| -Can produce proteins in plants
87
Advantages of producing proteins using plants
- Simple to grow
- Can grow in large bioreactors
- Cheaper than E. coli
- No ethical issues
- Safe
- Glycosylates secreted proteins
88
Disadvantages of using plants to produce proteins
- Codon usage differs (codon bias)
- Slow growth
- Highly regulated: laws against using plant cells
89
Features of model insect Drosophila melangoster
2-4 mm long
Easy to maintain
A model organism for studying fundamentals of eukaryotic genetic regulation and developmental biology
2-week life cycle
Diverged from humans 600 million years ago
12,000 genes, 4 chromosomes
Many mutants available for research
90
What type of chromosomes does Drosophila have?
Polytene chromosomes
Chromosomes duplicate to increase protein production but don’t divide
Very large - easily viewed by light microscope
Characteristic banding pattern
91
Advantages of using insects for recombinant protein production
- Growth media relatively simple
- Relatively cheap
- More robust than mammalian cells
- Safe
- Effective post-transcriptional modification
92
Disadvantages of using insects for recombinant protein production
- More expensive than using E. coli/yeast
- More complex than E. coli/yeast
- Lower yields than E. coli/yeast
93
What is an example of an insect vector?
Baculoviruses
94
What are baculoviruses?
- Only infect insects, arachnids and crustaceans
- They cause wilting disease in larvae producing silk
- Used as biopesticides in the 1940s
- 80-180kb
95
Process of infection of an insect by a baculovirus
1. Insect eats virus-contaminated leaf
2. In intestine epithelial cells, single virus particles are released that infect adjacent cells
3. Late infection: packages of virus particles are formed (polyhedrons)
4. Cells lyse and the polyhedrons are released. These protect the virus until it is infested by another insect
5. Makes insect climb as it does to release particles onto plant leaves
96
AcMNPV: the most common vector used in insect transformation
- Autographa californiaca multiple nuclear polyhedrosis virus
- Isolated from the species Autographa californica (moth)
- Used for complex proteins
97
What part of a baculovirus is used to create a transgenic insect?
The polyhedron gene has a strong promoter and this is exploited for transgenic expression
98
Producing the AcMNPV vector
1. E. coli transfer vector contains gene of interest. This contains the polyhedron promoter and flanking sequences from AcMNPV
2. This transfer vector is then transfected into insect cells with the AcMNPV vector
3. A double crossing over event generates the recombinant vector
99
What does transfect mean?
Infect a cell with free nucleic acid
100
Why are Bacmids useful when creating a transgenic insect?
- They replicate in E. coli and jnsect cells - shuttle vector
- Entire baculovirus, plus sections from an E. coli vector
- Construct plasmid with bacterial ORI, MCS and selectable marker, miniprep from E. coli and then transfect cells
101
How to make a transgenic insect
- Must affect the germline because animal cells are not totipotent
- Transposons known as P elements are found in Drosophila
- P-carrying flies also have a repressor, which means transposition frequency is very low
- P-negative flies have normal transposon activity
- If P-carriers are crossed with P-negative negative flies, transposon activity is briefly very high and transposons can be inserted due to a lack of repressor
- Produce helper plasmid with inverted repeats flanking the P element (one of which has partially deleted inverted repeats, so the transposon can’t move). Between the P elements is the transposase gene which is responsible for excising the region of DNA
- Produce a vector plasmid with your selected transgene, a selectable marker gene and intact invert repeats
- Inject both plasmids into an egg
- Some nuclei within the egg will be transformed, others will not
- Mate the adult that forms from this egg with a wild type fly
- Some of the offspring will carry the transgene
102
What does Zika virus do?
Cause microcephaly - where the head circumference is smaller than normal
103
What is a rosy- mutant Drosophila?
A mutant fly with brown eyes (wild type has red eyes)
104
What causes Dengue fever?
A virus
105
What parasite causes malaria? What carries it?
Caused by parasite called Plasmodium sp.
| Carried by mosquito - Anopheles gambiae
106
What is used for screening when creating transgenic mosquitos?
GFP or eye colour
107
How can mosquitos be engineered to prevent them spreading malaria?
They can be engineered to be less susceptible to Plasmodium by increasing levels of immune system protein defensin A, or express bee venom phospholipase in mid-gut
108
How to replace wild mosquito population with GM one
- Use two genes, A and B, which must both be inherited together for survival
- These genes are linked to insecticide susceptibility genes
1. Engineer males homozygous for A and B
2. Release into the wild
3. Mate with wild females
4. These hybrids will each have one copy of A and B and survive
5. Hybrids go on to mate with wild mosquitos and offspring that don’t inherit A and B together die
109
Features of model worm: Caenorhabditis elegans
- 1mm long
- Feeds in bacteria, lives in soil
- Two sexes - hermaphrodite and male
- Simple, non-segmented tube body
- Cuticle later to prevent dehydration
- Sense organs for taste, smell, touch and temperature
- No eyes
- Nerve ring and nerve cord
- 3 day life cycle
- Nerve ring and nerve cord
- 4 larval stages
- Easily maintained in lab
- Transparent so can easily see fluorescent proteins being expressed
110
How many cells does C. elegans have?
959 - precise number
111
Other names for gene silencing
- Quelling
- Post transcriptional Gene Silencing (PTGS)
- RNA interference (RNAi)
112
How was gene silencing first discovered?
- Fire and Mello were investigating how gene expression was regulated
- dsRNA added to culture medium and taken up into cells of C. elegans
- Or RNA could be microinjected into eggs
- Injected ‘sense’ mRNA encoding a muscle protein, but this led to no changes in the behaviour of worms
- Injected ‘antisense’ mRNA, but also had no effect
- Twitching movements observed when two types of RNA combined to produce dsRNA - this movement would also be observed if the worms didn’t have the gene for the muscle protein - it had been switched off
113
How is Flavr Savr tomato produced?
Used Agrobacterium tumefaciens to introduce an antisense copy of the polygalacturonase gene
Delays ripening and prolonged shelf life
114
How does gene silencing work?
- Sense and antisense strand produced separately and they anneal to produce double-stranded mRNA - not translated if ds
- Depends on the organism
- With C. elegans is via RNA injection or feeding
- With most other species, you transform your target organism with a plasmid that contains your construct so antisense mRNA can be produced and anneal to complementary sense RNA
- You can have a dual promoter, so have a gene read in both directions using a different promoter on each side. Only one vector needed
- Hairpins can be used to make double-stranded mRNA. Promoter -> sense ORF-> intron -> antisense ORF -> Terminator. DNA transcribed in loop (loop is intron)
115
Why does the introduction of additional RNAs silence genes?
- Aside from protein coding, RNAs have many other cellular functions
- Large RNA molecules can block translation by preventing ribosome binding or block post-transcriptional modification by preventing binding of the spliceosome
116
What is the main purpose of RNAi in cells?
RNA interference is the main viral defence in eukaryotes - many viruses consist of dsRNA
117
Outline the process of RNAi gene silencing
1. Initiation phase: Dicer enzyme recognises dsRNA and cleaves it into siRNAs 21-23 bp long
2. Effector phase: RISC unwinds siRNA, which can them bind to complementary mRNA
3. RISC and siRNA are now bound to complementary mRNA and Argonaut cleaves the mRNA, which is then degraded by exonucleases in the cytoplasm
4. RdRP synthesises more dsRNA from the RNA fragments, which are cut into more siRNAs by Dicer
5. siRNAs go on to activate more RISC to degrade more mRNA
- RNA can permanently repress gene expression - useful for generating mutants
- RISC and siRNA can ask bind to complementary DNA
- siRNA can direct heterochromatin-forming enzymes to the target gene location on the chromosome
- Once the open DNA is converted into heterochromatin, no more mRNA will be produced
118
Example of RNA silencing: potato
- Potatoes contain toxic steroidal glycoalkaloids (SGAs) e.g. solanine
- Cooking at high temperatures produces acrylamide, a carcinogen
- RNAi silencing has been used to inhibit a key enzyme in SGAs biosynthesis to reduce solanine
- The innate potato has reduced acrylamide due to silencing of asparagine synthetase (asn1) which makes the asparagine precursor
- Reduced browning due to reduction of ppo5 (polyphenol oxidase-5)
119
Outline the features of the model frog
- African clawed frog (Xenopus laevis)
- Long-lived: 15 yrs in wild, 30 in captivity
- First vertebrate to be cloned
- 23,000 genes
120
Outline the features of the model fish
- Zebrafish (Danio rerio)
- 200 eggs laid and transparent embryos develop in a few days
- 4cm long
- Genome 1700 Mb
- 25 chromosome pairs
- 450 My divergence from humans
- Shares 70% of human genes
- Used in developmental studies, immune system studies, circadian biology etc
121
Outline the mammal model organism
- Mouse (Mus musculus)
- Most closely related model organism to humans
- 2500 Mb genome
- 99% of genes have human orthologs
- Fast lifecycle: 8 litters per year
- 2 year life cycle
- Transgenic knockout lines to find gene function
122
Advantages of recombinant protein production in mammalian cells
- Effective post-translational modification
- Accurate folding
- Codon usage correct
- Intron splicing accurate
- Little regulation required
123
Disadvantages of recombinant protein production in mammalian cells
- Difficult to culture cells
- Expensive
- Slow
- Limited large scale production
- Low yield
- Susceptible to human viruses so less safe - must be kept in sterile conditions
124
What percentage of medical use recombinant proteins are produced using mammalian cell lines?
70%
125
What is the shuttle vector when transforming mammalian cells?
```
SV40 virus
Has E. coli and mammalian ORIs
Selectable marker for E. coli and mammalian cells
MCS
Mammalian promoter
3’ polyA tail
```
126
What selectable markers can be used on mammalian cells?
- There are very few antibiotics that can be used for mammalian cells
- Can use genticin (G-148) - similar to kanamycin and neomycin, prevents translation of proteins
- Selection can also be autotrophic: cells lacking dihydrofolate reductase (DHFR) gene are transformed with a plasmid encoding for the DHFR gene. Only transformed cells grow
127
Why are mammalian vectors more complicated? What is the solution to the problem?
- The proteins being encoded are often more complex, and several peptides must be made and assembled
1. Could use two vectors to express the subunits in one cell
2. Could use one vector with two genes under the control of different promoters
128
Selective breeding: what is it?
- Manipulation of animal genetics
| - Artificial insemination/liquid nitrogen/IVF used to breed selected individuals
129
What is cloning?
Generating progeny that are identical to the parent
130
What is transgenesis in terms of making a transgenic animal?
Creating an animal that involves the introduction of foreign DNA
131
What are totipotent cells?
Cells that can differentiate into all cell lineages
132
What are pluripotent cells?
Capable of forming all cell lineages within an embryo, but not extra-embryonic lineages. Embryonic stem cells are pluripotent
133
What are multipotent cells?
Cells that can differentiate into many, but not all, cell lineages
134
When is an embryo considered totipotent?
Only for the first couple of cell divisions
135
Steps of cloning frogs
Nuclear transplantation:
1. Remove nucleus from an egg
2. Add a somatic (from body cell) nucleus
3. Allow to develop into frog
- Showed that a nucleus from a differentiated cell can regenerate an entire organism
- Avoids ethical issue of using embryos
136
Why is it so much harder to clone mammals than amphibians?
Their eggs are 4000 times smaller
137
3 methods of forming a transgenic animal
1. Microinjection of DNA into fertilised eggs
2. Transform embryonic stem cells
3. Use a retrovirus
138
Step 1 of producing a transgenic animal: Microinjectjon of DNA into fertilised eggs
- Transgene is injected into male nucleus just after fertilisation
- No vector required
- This requires specialised equipment and a lot of skill
- Success rate is 5-40%
- Imtegration is random
1. Transgene injected into fertilised egg cells
2. Culture in vivo for a few days before implantation
3. Implant into foster mother
4. Some have stable transgene and in others it is lost. Founders will be heterozygous
5. Cross two heterozygous founders and get 25% homozygous progeny
139
Method 2 of producing a transgenic animal: transform embryonic stem cells
- Stem cells derived from the blastocyst and can differentiate into any body cell
- They can be cultured as per any cell line
- Embryonic stem cells do not age as normal cells do
- Ethical issues
- Progeny are CHIMERIC as transgene only integrated into some cells
- Stem cells from agouti mice are usually used as agouti is the dominant coat colour
1. Make vector containing gene of interest
2. Transform stem cells from agouti mouse
3. Insert stem cells into embryo from white mouse
4. Implant into mouse
5. Founder offspring are chimeric
6. Cross founder with white female - any agouti offspring have transgene
140
Advantages of microinjection of DNA into fertilised egg cells method
- Easier in large eggs
| - Few chimeras - individual only has one cell lineage
141
Where can embryonic stem cells be harvested from?
- Surplus embryos from IVF
- Aborted foetus
- Therapeutic cloning - transfer of a nucleus into an enucleated embryo
142
What are iPSCs?
induced pluripotent stem cells - making stem cells from somatic cells
143
How can the method of transforming embryonic stem cells to create a transgenic mammal allow targeted introgression?
Knockout mice can be made to determine gene function - analogues to human genes
144
How to use a transgenic animal with knockout genes to analyse gene function:
1. Clone the regions flanking the gene into a vector with the gene replaced with a selectable marker
2. Introduce DNA construct into mouse
3. Construct replaces or disrupts the gene via homologous recombination, so no protein is made and gene function can be analysed
145
Method 3 of producing a transgenic animal: Use a retrovirus
- Only a single copy will be integrated into the genome
- No microinjection needed
- The retrovirus is added to the developing embryo and will infect it
- Sometimes viral DNA integrates as well as the gene of interest
- Chimeras are always produced because integration occurs after nuclear fusion
- Only small amount of DNA up to 8 kb can be integrated
146
Transgenic mice example
Rat somatotropin gene
Growth hormone from rats
Produces larger mice
This was the first case of stable gene inheritance and normal function
147
Bovine growth hormone example
- Recombinant bovine somatotropin (rBST)
- Cow BST cloned and expressed in E. coli
- Injected into cows to cause increased milk production
148
Transgenic fish example: salmon
AquAdvantage Atlantic salmon has a gene for a growth hormone from Pacific Chinook salmon and a promoter from an ocean pout
Salmon now grows all year instead of only in spring and summer
Reaches market size in half the time
149
What is the relationship between gene size and chance of random mutation?
The larger the gene, the bigger the target for random mutation
150
How to supplement a replacement gene for a defective gene in an organ through gene therapy:
1. Identification and characterisation of gene
2. Clone gene into vector
3. Deliver DNA into cells
4. Express replacement gene
151
How to get replacement gene in gene therapy into cells?
- Direct injection
- Aerosol inhalation
- Cell removal, treatment and implantation
152
What is the most common form of vector used in gene therapy?
- Virus vectors
- 70% of gene therapy uses them
- Adenoviruses or retroviruses
- Most common other ways are DNA, RNA or liposomes
153
Features of Adenovirus
- Infect vertebrates and humans
- Cause respiratory diseases
- dsDNA virus, 36 kb
- Icosahedral capsule (20 faces)
- Pentons every 5 faces and these consist of a pentamer base and a fibre
- The pentons bind to the host cell
154
Steps of Adenovirus infection
1. Adenovirus fibre tip binds to CAR receptor found in many tissues
2. Penton base binds to integrins on host cell wall
3. Membrane forms vesicle and takes virus in
4. Virus is released into the cytoplasm and moves towards the nucleus
5. The virus disassembles and DNA enters the nucleus
155
Advantages of using adenoviruses as vectors
- Relatively harmless, do not cause rumours
- Easy to culture and produce in large amounts
- Life cycle and basic biology understood
- Function of viral genes is known
- Genome sequence available
156
Disadvantages of using adenoviruses as vectors
- Limited by size
- Virus is short-lived (2 weeks)
- Immunity develops, can’t reinfect
157
What must be deleted from the adenovirus to use it as a vector?
The EA1 gene so the virus can not replicate
158
What causes cystic fibrosis?
- Homozygous recessive mutation in the cystic fibrosis transporter gene (cftr)
- Massive gene, only 2% exons
- 3 nucleotide deletion at 508 AA position is the cause in 70% of US sufferers
- The other 30% of cases caused by other mutations
159
What are the symptoms of cystic fibrosis?
- In healthy cells, the CFTR protein forms an ion channel that opens and closes as needed as a phosphate group attaches and detached
- In CF sufferers, defects in the channel lead to thickening of the mucus that coats the lungs
- Obstruction, pathogen growth, scar tissue and respiratory failure
- Life expectancy is 40-50 years
160
How was gene therapy previously used to treat cystic fibrosis?
- First tested in rats, then humans
- Healthy cftr was cloned into adenovirus vector and then inhaled through nose
- Gene was expressed in lungs and normal ion channel function was restored
- Effect was temporary
- Issues with severe immune responses led to a patient death in 1999, so adenoviruses are no longer used to treat CF
161
What is a liposome?
A minute sac of phospholipid molecules enclosing a water droplet, especially as formed artificially to carry drugs or other substances into the tissues
162
What are the methods of gene therapy used against cancer?
1. Direct replacement: add functional gene
2. Direct attack: tumour necrosis factor
3. Suicide: gene and anti-cancer therapy
4. Immune provocation: cell membrane proteins
163
Features of retroviruses
- Infect mammals, need dividing cells
- Various diseases including cancer and HIV caused by retroviruses
- Positive sense ssRNA, 7-10 kb
- Protein capsid and envelope from the plasma membrane of previous host cell
- A few molecules of reverse transcriptase
164
Process of retrovirus infection
1. Cell infected by retrovirus
2. RNA is copied into DNA by reverse transcriptase
3. DNA circularises and is integrated into the host genome
4. A strong promoter in the LTR region enables transcription of the viral genes
5. New virus particles are then assembled and packaged and envelope glycoproteins are acquired from the host cell membrane
165
Example of using a retrovirus as a vector
- Murine leukaemia virus (MuLV)
- Vectors have all viral genes removed so just have LTRs and packaging signal
- Can insert 6-8 kb
- This is integrated into the DNA of the producer cell
- The virus particles only contain the vector with the cloned gene insert and molecules of reverse transcriptase
- In the cell, RNA is transcribed into DNA and DMA integrated into the genome
- No immune responses seen
- Permanent effect
166
What is SCID?
- Severe combined immune deficiency
- Disease where B and T cells of the immune system are defective
- An illness could be fatal
- 25% of cases due to mutation in ada gene, responsible for producing adenosine deaminase and involved in lymphocyte production
- Gene therapy could be hard to add functional copies of ada gene
167
Process of adding functional copy of ada gene to child with SCID
1. Add ada gene into vector with selectable marker
2. Isolate bone marrow stem cells
3. Culture cells in vitro
4. Infect with retrovirus vector
5. Transfer cells back to body
6. lymphocytes with functioning ada gene now made
168
Result of treating SCID with gene therapy
- Tested since 1991
- Also treated with enzyme replacement therapy
- 100% survival rates reported with 75% of children needing no further enzyme treatment
169
What are AAVs?
Adeno-associated viruses
Satellite virus - needs another virus to work
ssDNA, 5 kb
What is used in most new clinical trials as a vector
170
Advantages of using AAV vectors for gene therapy
- No immune system response
- No inflammation in the body
- Affects vertebrates
- Works in non-dividing cells
- Integrates in a specific site in the genome
171
Disadvantage of using AAV vectors for gene therapy
Small so can only package up to 5 kb
172
What conditions have AAVs been used to treat?
Childhood blindness due to defect in retinal pigment
Haemophilia
Muscular dystrophy
173
Example of non viral delivery in gene therapy: liposomes
- Used in 10% of gene therapy trials
- Add DNA into hollow liposome; liposome will fuse with the plasmid membrane; DNA will be delivered into host cell (lipofection)
- Non-specific delivery
- Can inject into cancer cells
174
Example of non viral delivery in gene therapy: RNA
- Several techniques including antisense and RNAi
- Can use liposomes to deliver RNA
- Or can express DNA to be transcribed into antisense RNA
- Antisense RNA has been used with malignant glioma brain cancer cells in rat trials - antisense IGF1 stooped tumour growth caused by gene IGF1
- RNAi most frequently used RNA-based gene therapy
- Can inject siRNA into the eye directly to target age-related macular degradation
- No siRNAs have been approved for clinical use
