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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
Q

How do we localise a gene of interests encoded protein?

A

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
Q

What is EDMD?

A

Emery-Dreifuss Muscular dystrophy

Lots of mutations in Lamin A C2C12 myoblasts ( nuclear envelope protein)

27
Q

How do we localise the EDMD protein?

A

Transfect Lamin A cell types
Tag lamin A protein
Lamin a expressed throughout cell nucleus, throughout nuclear envelope
Lamin A localised in foci

28
Q

What effect does transfecting mutations have on patients?

A

However transfecting mutations seen in the patients causes lamin A protein to localise in these strange foci
Variants cause dysfunctional protein behaviour

29
Q

What are IPSCs?

A

Induced pluripotent stem cells are fibroblasts that are genetically reprogrammed embryonic stem cells

30
Q

What gene editing tools are available?

A

CRISPR and Taler

31
Q

How is CRISPR used to aid patients with Duchenne-muscular dystrophy (DMD)?

A
  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
Q

Why is cell culturing not enough to diagnose?

A

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
Q

What alternative to cell culturing is often used ?

A

90% use animal research

34
Q

How has animal research contributed to healthcare?

A

Most medicines used today come from animal research
Contributed to 70% nobel prizes
Aided cure for Polio

35
Q

What is ASPA?

A 
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
Q

What requirements need to be met in order to qualify for animal use?

A
  • 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
Q

Why are mice the ‘go to’ research animal?

A

< genetically similar to humans as mammals

Been used in biomedical science for years

Has accelerated lifespan

38
Q

How is a mutant mouse created?

A
  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
Q

What is the significance of zebrafish in genetic disease?

A

vertebrate Model for Human Genetic Disease

40
Q

How are zebrafish used in gene knockdown experiments?

A

MO blocks gene specific translation or splicing

  • Translation Blocking MO’s
  • Splicing inhibiting MO’s
41
Q

What is MO?

A

Morpholino oligonucleotides (MO) with a morpholine backbone

42
Q

Why are zebrafish MO used in knockdown experiments?

A

MO backbones v. stable and undergo complementary base pairing
Can synthesise MO to target specific genes

43
Q

What are the products of using zebrafish MO?

A

Can have off-target genetic effects

A mutant is the gold standard

44
Q

How are zebrafish mutants formed?

A

Can mutenise fish by bathing them in mutagens e.g. ENU

45
Q

What is EMU?

A

Potent mutagen targeting spermatogonial stem cells

Causes point mutations at a rate of 1 per 700 gametes

46
Q

What are the 2 major ENU mutants formed?

A
  • Forward genetics (phenotype based)

- Reverse genetics (genotype based)

47
Q

Describe the outcome of forward genetics

A

ENU Screening (phenotype based) - obvious changes e.g. lacking eyes (masterblind mutation)

48
Q

Outline effects of reverse genetics

A 
ENU Screening (Genotype based)
F1 genomic DNA exome screened or sperm cryopreserved
49
Q

What is CRISPR?

A

Clustered regularly interspaced short palindromic repeats (CRISPR)

50
Q

What is Cas9?

A

CRISPR associated protein 9 (Cas9)

Bacterial adaptive immune system

51
Q

What is PAM?

A

Protospacer (target sequence of guide RNA) - protospacer adjacent Motif

52
Q

How does Cas9, CRISPR and PAM work?

A

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
Q

What is the role of RNA Rescue experiments?

A

> proving pathogenesis

54
Q

What is ARCAs?

A

Autosomal recessive cerebellar ataxias is a neurodegenerative disorder

55
Q

How is RNA rescue used for ARCAs patients?

A

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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