WEEK 11: MODEL ORGANISMS AND GENE TECHNOLOGIES Flashcards
1
Q
What is forward genetics?
A
- Looking at the phenotype and moving to genotype
- e.g. biochem function of protein, mapping, sequencing, linkage, new understanding of genetics (e.g. X linked traits)
- Genome analysis –> new identified gene from mapping and genome sequencing
- No known function
2
Q
What is reverse genetics?
A
- Starting with the gene and observing the phenotype
- e.g. Model organisms, GMOs, Transgenic organism –> TO phenotype like disease in humans or flower colour
3
Q
What does the branch length value give in phylogeny trees?
A
- Gives the fraction of sequence substitutions i.e. 0.1= 10 substitutions per 100 residues
4
Q
What is CRISPR/Cas9 more efficient for targeting?
A
- More efficient for targeting two different sites in a gene
5
Q
to make transgenic mice for OVEREPXRESSING a gene, which step can they skip?
A
- Can skip the ES cell step
6
Q
What is the difference between orthologues and paralogues?
A
- Orthologues= homologues (genes/proteins) found in different species
- Paralogues= Homologues found within the SAME species
7
Q
What do humans and mice have in common?
A
- Adaptive immune system (produce Igs)
- Similar anatomy
- Nervous system (including behavioural studies)
- Warm blooded/similar metabolism/urea excretion
- Gold Standard
8
Q
What do humans and zebrafish have in common?
A
- Both vertebrates/bony organisms
- Adaptive immune system (Igs)
- Embryo development
- Circulatory system just like mammals
9
Q
What do humans and worms & flies have in common?
A
- Bilateria clade of animals
- Blastocyst formation
- Motile
- Basic muscle, nerve, and GI similarities
- Three germ layers (eto, medo, endoderm)
- Several aspects of innate immunity shared
- Cell-cell signalling pathways conserved
10
Q
What do humans and yeast have in common?
A
- Eukaryota
- Similar organelle position
- Similar metabolism
- Study of transcription/translation /gene regulation/cell cycle
11
Q
What is so good about using the mouse as a model organism?
A
- 90% of mouse and human genomes syntenic
- At nucleotide level, 40% of human genome can be aligned with mouse
- Non-gene features are conserved and important
- Has about 30,000 genes (orthologues 80%)
- 80 000 SNPs already identified
- High relevance to human health
12
Q
What isn’t so good about using the mouse as a model organism?
A
- Expensive to maintain
- Expensive to genetically manipulate
- Need ethics approval for ALLLL experiments!
- Bodies aren’t transparent
- Embryos inside the body
- Low progeny number
- Time consuming
13
Q
What does syntenic mean?
A
- E.g. Human chromosome 1 is in mouse chromosomes 1 and 4
14
Q
When are rats used instead of mice?
A
- For physiology/pharmacology research because their physiology is much closer –> complex diseases such as Hypertension and metabolic diseases
- Their genome has been sequenced
- Gene knockouts can be used (CRISPR) but not all the time
15
Q
Why are mice better than rats for genetics?
A
- Ease of gene targeting by homol. recombination
16
Q
What is so good about using the zebrafish as a model organism?
A
- Transparent body of embryos and larvae; real-time mapping of early development
- Stock centres
- Drug screening in 96 well format
- Live tracking for behavioural studies
- Embryo manipulation is relatively easy
- Faster replicating/ more progeny than mice
17
Q
What isn’t so good about using the Zebrafiash as a model organism?
A
- Traditional reliance on morpholinos ; transient knockdown
- Gene targeting by CRiSPR/Cas9 becoming more routine
- Less ‘translatable’ than mice
- Needs ethics approval: vertebrates
- Fish genomes have MASSIVE duplication events that can complicate looking at orthologues –> must knock down 2 orthologues in zebra fish to = the 1 in humans
- Requires specific, expensive infrastructure
18
Q
What is so good about using Drosophila?
A
- Huge range of genetic tools available
- Excellent dev. biology model
- 75% of human disease genes have a fly homologue
- Cheap, fast replicating (10 days), many progeny
- Non vertebrate = NO ETHICS APROVAL!
- Many phenotypic tests: behaviour, toxicology
- Whole genome forward genetics screens: RNAi
- Drug screening in 96 well format
19
Q
What isn’t so good about using Drosophila?
A
- It is not a vertebrate, nor a deuterostome
- No crypreservation
- Limited antibodies available but reporter gene libraries (in vivo constructs that tell you where the gene is like GFP )
- Very small tissues; biochem difficult
- No adaptive immune system
- Less ‘translatable’ than zebrafish
- Not fully transparent
- PC2 rather than PC1 like mice,worms
20
Q
What is really good about using C.elegans as a model organism?
A
- 42% of disease genes have worm homologues
- Gene manipulation EASILY performed
- Completely transparent and all 959 somatic cells fate mapped
- Quicker (3.5 days), cheaper, smaller
- NO ETHICS REQUIRED!
- Mutants easily stored and regenerated
- Whole genome forward genetic screens: RNAi
- Drug screening in 96 well format
21
Q
What isn’t so good about using C.elegans as a model organism?
A
- Evolutionary more distant (not a deuterostome)
- Gene targeting is difficult (libraries of mutated strains are used)
- Organ development different to humans
- Lack key cell-cell signalling pathways
- Limited antibodies available
- Very small tissues; biochem difficult
- No adaptive immune system
22
Q
What is good about using S.cervisae (aka yeast) as a model organism?
A
- Single cell eukaryote (fungi)
- Genetic analysis, manipulation easy (library of deletion mutants)
- Cryopreservation easy
- Super cheap, super fast, super compact
- Normally studies in the haploid state
- High throughput methods available
- Essential cellular processes: DNA repair, Cell cycle checkpoint, Mitochondrial research, Organelle, protein processing and secretion
23
Q
What isn’t so good about using yeast as a model organism?
A
- Evolutionary distant (20% of human genes have yeast orthologues)
- Limited to single cell phenotypes (hard to relate to multicellular organisms just like humans)
24
Q
What is good about using E.coli for research?
A
- Ultra cheap, fast, ultra compact
- Normally stuided in haploid state
- High throughput methods available
- Great for producing large amounts of protein for structural studies
25
What is not good about using E.coli for research?
- Prokaryote
| - Limited to single cell phenotypes
26
out of all the model organisms, which is the most expensive?
- Mouse
27
Out of all the model organisms, which has the highest birth rate/progeny?
- The worm!!
28
Out of all the model organisms, which one has the shortest and longest generation time respectively?
- Yeast and Mouse
29
Out of the model organisms, which ones need ethics approval?
- Zebrafish and mouse (vertebrates)
30
Which model organisms can be frozen (storage)?
- Yeast
- Worm
- Zebrafish
- Mouse
(pretty much all of them except flies)
31
Which of the model organisms has the most and least genomic homology to humans respectively (also phenotype relationship)?
- Yeast and mouse
32
Can gene targeting be performed in all of the model organisms?
- YES!
33
What is the process of random mutagenesis to investigate biological processes?
- WT animals--> Mutagenise--> Select for mutants defective in biological process e.g. patterning, cell growth--> Identify mutated genes mapping/sequencing --> Determine role of genes in biological processes
34
Which 3 forms of mutagenesis are performed on Drosophila?
- X-rays
- EMS
- P elements
35
What are morpholinos?
- Synthetic molecules that bind and are complementary DNA of interest
36
What are the two main methods for gene knockdown?
- RNAi
| - Morpholinos
37
What is good about using RNAi and morpholinos?
- Can target ALL genes in a genome
- Can target genes in specific cells or tissues
- Variable knockdown efficiency --> allelic series, avoid animal/cell lethality with hypomorphs (if you knockdown too much you can kill it)
38
How is gene knockout performed in mice?
- Homologous recombination via ES cells
| - Completely knocks out the gene for 0% function
39
What is the process for gene knockout in mice?
1) Select for neomycin resistance --> take all of the ES cells that are NeoR and make a COPY of them
2) Test the copy for thyamine kinase (TK)
3) If TK is +ve-->THROW IT AWAY because they are random intrigants
4) If TK is -ve--> KEEP IT!! Because this means homologous recombination has occurred.
40
What is the CRE/LoxP system (borrowed from yeast) used for?
- For genes that cause death when knocked out (i.e. essential genes)
- It takes a few generations
41
how do you validate gene knockout methods?
-Via a Western Blot to see if the protein is present
42
Is the spatial and temporal specificity obtained with an inducible Cre/LoxP system?
- YES!
43
What happens if in CRISPR/Cas9, the PAM sequence is not present?
- There will be no cleavage
44
In CRISPR/Cas9, what does the SgRNA do?
- Targets the gene of interest (complementary to the target sequence)
45
In CRISPR/Cas9, what is the precise repair of the cut due to?
- Homologous recombination
46
Which 3 processes can result from CRISPR/Cas9 technology in mice?
- Knockout mice
- Single aa substituted mice
- Floxed mice
47
What is really good about CRISPR/Cas9?
- Can target any genes in a genome
- Quick, relatively cheap
- Works in all organisms tested
- Can target genes in specific cells/tissues
- Can control temporally/spatially
48
What isn't so good about CRISPR/Cas9?
- Doesn’t always give desired alteration (need a good screening method)
- Off-target effects--> Need to re-sequence whole genome
49
To make transgenic mice for overexpressing a gene, which step do we skip?
- The ES cell step
50
What does the GAL4 UAS system allow for in Drosophila?
- To know whether the gene has been epxressed or not; when GAL4 has been epxressed, the gene will be expressed
