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Genomics > The Functional Genome > Flashcards

The Functional Genome Flashcards

1
Q

What is meant by the functional genome?

A

Describe gene (and protein) functions and interactions

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2
Q

What is the significance of the functional genome?

A

The pipeline to genetic diagnosis: proving a variant/mutation is pathogenic

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3
Q

Give examples of In Vitro techniques for studying gene (dys)function

A 
Cell culture
SiRNA, 
ShRNA
IPSCs
CRISPR
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4
Q

What are some of the in vivo methods used for studying gene (dys)function

A 
Mouse
Zebrafish 
ENU screens
Morpholinos
CRISPR
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5
Q

What is the significance of NGS methods like WES and WGS

A

They’re rapid modern methods for high throughput DNA sequencing

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6
Q

What is the use of WES?

A

used to capture the sequence of the coding region of the genome

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7
Q

What is WES?

A

Whole Exome Sequencing

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8
Q

What is WGS?

A

Whole Genome Sequencing

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9
Q

What is the purpose of WGS?

A

captures the whole genome - not always necessary

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10
Q

What is the aim of NGS WES and WGS?

A

These both aim to identify potential disease causing genetic variants; personalised medicine

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11
Q

Why is WES a useful gene filtering tool?

A

WES data is subjected to a prioritisation filtering protocol

15-20,000 coding SNPs reduced to one or several candidate genes

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12
Q

How are genes filtered using WES?

A

They’re checked for co-segregation (family members) and validated by Sanger sequencing

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13
Q

Why is WES gene filtering not enough evidence to prove a gene causes a disease?

A

Filtered WES doesn’t prove causality

need further functional evidence

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14
Q

What other evidence is needed to prove causality after WES filtering?

A

Need to assess how the variant affects:

  • tissue / cell expression
  • knockdown / over-expression => affects phenotype
  • protein detection in patient samples
  • protein behaviour
  • cell / tissue development
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15
Q

How can we assess if a patient’s protein is affected?

A

Blood or tissue biopsies

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16
Q

Which mutation is commonly tested for using biopsies?

A

Identification of mutations in MYL1 in patients with congenital muscular dystrophy by WES

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17
Q

What is the function of the MYL1 gene?

A

Gene expressed in fast twitch muscle, patients have reduced fast muscle fibres

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18
Q

What is the downfall of using biopsies to diagnose gene variant causing disease?

A

GOI will not always be expressed in the blood and might not be in an accessible affected tissue

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19
Q

How can we culture animal cells in vitro ?

A

Removal of cells from an animal and subsequent growth in favourable conditions

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20
Q

Why are primary cells used for in vitro cell culturing?

A

Primary cells have finite divisions but can immortalise to provide a continuous source
Provides a cheap, rapid and reproducible model for studying the normal physiology and biochemistry of cells

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21
Q

What alternative is there for animal models?

A

A good alternative to using animal models, reducing numbers of animals being used in research, less restrictions
Many tissue specific cell lines commercially available

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22
Q

What is gene knockdown?

A

RNAi mediated gene silencing
Based on endogenous microRNA gene silencing
Modified to include GOI complementary sequence

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23
Q

What is ShRNA?

A

Short Hairpin RNA (ShRNA)

24
Q

Outline how RNAi mediated gene silencing (knockdown) occurs?

A

Packaged in a DNA plasmid, expression controlled by a RNA Polymerase III promoter

50-70nt. Exits nucleus, cleaved by a nuclease called Dicer (cytoplasm)

Cleaved segments bind to RNA induced silencing Complex (RISC) and direct cleavage and degradation of complementary mRNA

Short interfering RNA (SiRNA); similar to ShRNA, chemically synthesised, not vector based

25
How do we localise a gene of interests encoded protein?
Antibody staining - protein of interest - downstream target Transfect cells with GFP tagged GOI - (CMV Promoter) Transfect cells ith GFP tagged mutated GOI - (CMV Promoter)
26
What is EDMD?
Emery-Dreifuss Muscular dystrophy | Lots of mutations in Lamin A C2C12 myoblasts ( nuclear envelope protein)
27
How do we localise the EDMD protein?
Transfect Lamin A cell types Tag lamin A protein Lamin a expressed throughout cell nucleus, throughout nuclear envelope Lamin A localised in foci
28
What effect does transfecting mutations have on patients?
However transfecting mutations seen in the patients causes lamin A protein to localise in these strange foci Variants cause dysfunctional protein behaviour
29
What are IPSCs?
Induced pluripotent stem cells are fibroblasts that are genetically reprogrammed embryonic stem cells
30
What gene editing tools are available?
CRISPR and Taler
31
How is CRISPR used to aid patients with Duchenne-muscular dystrophy (DMD)?
1. DMD patient-derived IPSCs (lack exon44 & 45) 2. Genome editing by Taler / CRISPR 3. Changes DNA via exon skipping 4. Translated DNA is in-frame but exons are (44 & 45) repeated 5. Produces a truncated but functional dystrophin protein
32
Why is cell culturing not enough to diagnose?
Cells behave differently in a petri dish/flask compared to how they behave in a whole organism: 2D vs 3D Doesn’t replicate the actual conditions inside an organism or signals from other tissues No information about gene expression and function, with regards to developmental phenotypes
33
What alternative to cell culturing is often used ?
90% use animal research
34
How has animal research contributed to healthcare?
Most medicines used today come from animal research Contributed to 70% nobel prizes Aided cure for Polio
35
What is ASPA?
``` The animals (Scientific Procedures) Act (ASPA) - regulates use of protected animals in any experimental or other scientific procedure which may cause pain, suffering, distress or lasting harm to the animal ```
36
What requirements need to be met in order to qualify for animal use?
- Benefit > cost - No non-animal alternative available - Minimum no. of possible animals used - Animals with lowest pain sensitivity used - Pain is minimised - Research premises have necessary facilities to care for animals
37
Why are mice the 'go to' research animal?
< genetically similar to humans as mammals Been used in biomedical science for years Has accelerated lifespan
38
How is a mutant mouse created?
1. Targeting vector constructed 2. Introduced through homologous recombination into nucleus of pluripotent ES Cells 3. HR integrates in the cassette. Es selected based on ABr 4. +ve ES cells grown to blastocysts and implanted into pseudopregnant recipient mice
39
What is the significance of zebrafish in genetic disease?
vertebrate Model for Human Genetic Disease
40
How are zebrafish used in gene knockdown experiments?
MO blocks gene specific translation or splicing - Translation Blocking MO’s - Splicing inhibiting MO’s
41
What is MO?
Morpholino oligonucleotides (MO) with a morpholine backbone
42
Why are zebrafish MO used in knockdown experiments?
MO backbones v. stable and undergo complementary base pairing Can synthesise MO to target specific genes
43
What are the products of using zebrafish MO?
Can have off-target genetic effects | A mutant is the gold standard
44
How are zebrafish mutants formed?
Can mutenise fish by bathing them in mutagens e.g. ENU
45
What is EMU?
Potent mutagen targeting spermatogonial stem cells | Causes point mutations at a rate of 1 per 700 gametes
46
What are the 2 major ENU mutants formed?
- Forward genetics (phenotype based) | - Reverse genetics (genotype based)
47
Describe the outcome of forward genetics
ENU Screening (phenotype based) - obvious changes e.g. lacking eyes (masterblind mutation)
48
Outline effects of reverse genetics
``` ENU Screening (Genotype based) F1 genomic DNA exome screened or sperm cryopreserved ```
49
What is CRISPR?
Clustered regularly interspaced short palindromic repeats (CRISPR)
50
What is Cas9?
CRISPR associated protein 9 (Cas9) | Bacterial adaptive immune system
51
What is PAM?
Protospacer (target sequence of guide RNA) - protospacer adjacent Motif
52
How does Cas9, CRISPR and PAM work?
Guide RNA binds to strand of genomic DNA Cas9 endonuclease binds to non protospacer portion of gRNA + PAM of DNA DSB 3bp upstream of PAM Mutations can be introduced through NICJ and 11DR
53
What is the role of RNA Rescue experiments?
> proving pathogenesis
54
What is ARCAs?
Autosomal recessive cerebellar ataxias is a neurodegenerative disorder
55
How is RNA rescue used for ARCAs patients?
WES identified mutation CHP1 - injecting RNA for CHP1 you can rescue crossing over trajectory of the neurons - isn’t rescued to the same degree as using the patients - neuronal out-branches aren’t as convincing

Genomics (20 decks)

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