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BMS2042 > WEEK 11: MODEL ORGANISMS AND GENE TECHNOLOGIES > Flashcards

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
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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

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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
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4
Q

What is CRISPR/Cas9 more efficient for targeting?

A
  • More efficient for targeting two different sites in a gene
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5
Q

to make transgenic mice for OVEREPXRESSING a gene, which step can they skip?

A
  • Can skip the ES cell step
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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
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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
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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
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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
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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
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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
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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
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13
Q

What does syntenic mean?

A
  • E.g. Human chromosome 1 is in mouse chromosomes 1 and 4
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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
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15
Q

Why are mice better than rats for genetics?

A
  • Ease of gene targeting by homol. recombination
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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
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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
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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
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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
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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
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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
Q

What is not good about using E.coli for research?

A
  • Prokaryote

- Limited to single cell phenotypes

26
Q

out of all the model organisms, which is the most expensive?

A
  • Mouse
27
Q

Out of all the model organisms, which has the highest birth rate/progeny?

A
  • The worm!!
28
Q

Out of all the model organisms, which one has the shortest and longest generation time respectively?

A
  • Yeast and Mouse
29
Q

Out of the model organisms, which ones need ethics approval?

A
  • Zebrafish and mouse (vertebrates)
30
Q

Which model organisms can be frozen (storage)?

A
  • Yeast
  • Worm
  • Zebrafish
  • Mouse
    (pretty much all of them except flies)
31
Q

Which of the model organisms has the most and least genomic homology to humans respectively (also phenotype relationship)?

A
  • Yeast and mouse
32
Q

Can gene targeting be performed in all of the model organisms?

A
  • YES!
33
Q

What is the process of random mutagenesis to investigate biological processes?

A
  • 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
Q

Which 3 forms of mutagenesis are performed on Drosophila?

A
  • X-rays
  • EMS
  • P elements
35
Q

What are morpholinos?

A
  • Synthetic molecules that bind and are complementary DNA of interest
36
Q

What are the two main methods for gene knockdown?

A
  • RNAi

- Morpholinos

37
Q

What is good about using RNAi and morpholinos?

A
  • 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
Q

How is gene knockout performed in mice?

A
  • Homologous recombination via ES cells

- Completely knocks out the gene for 0% function

39
Q

What is the process for gene knockout in mice?

A

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
Q

What is the CRE/LoxP system (borrowed from yeast) used for?

A
  • For genes that cause death when knocked out (i.e. essential genes)
  • It takes a few generations
41
Q

how do you validate gene knockout methods?

A

-Via a Western Blot to see if the protein is present

42
Q

Is the spatial and temporal specificity obtained with an inducible Cre/LoxP system?

A
  • YES!
43
Q

What happens if in CRISPR/Cas9, the PAM sequence is not present?

A
  • There will be no cleavage
44
Q

In CRISPR/Cas9, what does the SgRNA do?

A
  • Targets the gene of interest (complementary to the target sequence)
45
Q

In CRISPR/Cas9, what is the precise repair of the cut due to?

A
  • Homologous recombination
46
Q

Which 3 processes can result from CRISPR/Cas9 technology in mice?

A
  • Knockout mice
  • Single aa substituted mice
  • Floxed mice
47
Q

What is really good about CRISPR/Cas9?

A
  • 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
Q

What isn’t so good about CRISPR/Cas9?

A
  • Doesn’t always give desired alteration (need a good screening method)
  • Off-target effects–> Need to re-sequence whole genome
49
Q

To make transgenic mice for overexpressing a gene, which step do we skip?

A
  • The ES cell step
50
Q

What does the GAL4 UAS system allow for in Drosophila?

A
  • To know whether the gene has been epxressed or not; when GAL4 has been epxressed, the gene will be expressed

BMS2042 (11 decks)

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