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Genomics > The Functional Genome > Flashcards

The Functional Genome Flashcards

1
Q

What is the prioritisation filtering protocol?

A

The protocol that WES data is subjected to. It reduces the candidate genes to find the region that might cause a disease. It filters out those that are already in the database or common variants. It also does this by looking at family members etc.

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2
Q

Why is the prioritisation filtering protocol important?

A

It is important for developing drugs for personalised medicine

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3
Q

Why is more functional evidence required?

A

WES does not prove causality

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4
Q

How is WES used in patients?

A

It can be used with blood or tissue samples to find out how proteins are affected. Some proteins will not be expressed in blood and this will show which genes are affected.

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5
Q

Why are cell culture techniques used?

A
  • In vitro (artifical environment)
  • Removal of cells from an animal and subsequent growth in favourable conditions
  • Primary cells have finite divisions but can immortalised to provide continuous source.
  • Provides a cheap, rapid and reproducible model for studying the normal physiology and biochemistry of cells
  • It is a good alternative to animal models so less restriction.
  • Many tissue specific cell lines commercially available - different tissue types such as neuronal and myogenic
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6
Q

What is gene knockdown?

A

microRNA based gene silencing technique

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7
Q

What is the microRNA based gene silencing technique?

A

microRNA adapted shRNA joins with a simple hairpin shRNA. This makes a protein which is exported out of the nucleus. It forms a mature microRNA duplex, then the mature microRNA which cleaves and does gene silencing.

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8
Q

What is a short hairpin RNA (shRNA)?

A
  • Based on endogenous microRNA gene silencing

- Modified to include GOI complementary sequence

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9
Q

Explain how a shRNA is used

A

Packaged in a DNA plasmid, expression is controlled by an RNA polymerase III promoter ubiquitously.
- 50-70nt. Transcribed. Exits nucleus, cleaves it through the nuclear pore called Expoitin-5; cleaved by a nuclease called Dicer in the cytoplasm
- Cleaved segments bind to RNA induced silencing complex (RISC) and direct cleavage and degradation
of complementary mRNA -> gene of interest.

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10
Q

What is short interfering RNA?

A

SiRNA similar to ShRNA, chemically synthesised, not vector based

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11
Q

Give an example of a shRNA

A

PDZRN3

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12
Q

Where is PDZRN3 found?

A

It is found to be developmentally regulated in skeletal muscle

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13
Q

What are C2C12 cells and what do they do?

A

They are immortalised mouse myoblasts that differentiate into muscle

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14
Q

What causes upregulation of PDZRN3 and MCH?

A

PDZRN3 blocks SiRNA upregulation of PDZRN3 and MHC.

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15
Q

What happens when PDZRN3 is knocked down?

A

Inhibition of myotube formation and MHC expression if it is knocked down, the myotubes don’t form.

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16
Q

How is the gene of interest encoded protein localised?

A

Through Antibody staining find the:

  • Protein of interest
  • Downstream target

Transfect DNA plasmid inserted into cell cells with GFP tagged GOI (CMV promoter)

Transfect cells with GFP tagged mutated GOI (CMV promoter)

17
Q

What is dysfunctional protein behaviour?

A

With the mutation, the protein is localised to specific areas

18
Q

Why is cell culture not enough?

A
  • Cells behave differently in a petri dish/flask to how they behave in a whole organism.
  • Doesn’t stimulate the actual conditions inside an organism.
  • No information about gene expression and function, with regards to developmental phenotypes
19
Q

What percentage of research is non-animal based?

A

90%

20
Q

Why are animals used in research and what are the benefits?

A
  • Cells behave differently in vitro to in vivo.
  • Most medicines we have today come from animal research
  • Contribute to 70% of the Nobel prizes
  • Polio
  • Anything found is also used in veterinary science for animal ill-health
  • Research scientist seek to alleviate pain and suffering
21
Q

Which animals are used in research?

A
  • 2.11M procedures on mice
  • 0.28m procedures on chicks
  • 4,481 procedures on dogs
  • 0.6m procedures on zebrafish
  • 0.32m procedures on rats
  • 10,424 procedures on horses
  • 159 procedures on cats
  • 3,207 procedures on monkeys
22
Q

What is the animals scientific procedures act (ASPA)?

A

Formed in 1986.

  • The UK parliament permitted the use of animals in scientific procedures.
  • it regulates the use of protected animals in any experimental or other scientific procedure that may cause pain, suffering, distress or lasting harm to the animal.
  • Animals cared for with the best standards of modern animal husbandry
23
Q

What are the animals protected under the ASPA 1986 act?

A

Any living vertebrae animal (other than man) and any living cephalopod (squid and octopus).

24
Q

Why are mice used as a mammalian model for human genetic disease?

A
  • Accelerated lifespan (1year = 30 human years)
  • Small
  • Reproduce quickly
  • Easy to handle and transport
  • More ethical than larger animals/non-human primates/humans
  • Modern genetic engineering have allowed for precise mutation to recreate a disease
  • Lots of mouse strains and models already exist
  • Mammals: genetically similar to humans
  • Mice have been used in BM research for 100+ years
25
Q

How is a mutant mouse made?

A
  • Targetted vector constructed and introduced into the nucleus of pluripotent ES cells
  • Take out the ES cell and select for the desired gene etc
  • HR integrates in the cassette. ES selected, based on AB.
  • Positive ES cells grown to blastocysts and implanted into a pseudo pregnant recipient mice.
26
Q

Disadvantage of mice experiments

A
  • Can get expensive
  • Have to develop in utero
  • Only produce on average 8 pups per litter which is low
27
Q

Describe the tissue specific KO mice experiement

A

The heart specific promoter is tissue specific.

When the genes are combined, the Cre will cut out the floxed gene and add Cre.

28
Q

What are zebrafish and why are the used in experiments?

A
  • Tropical freshwater fish
  • Transparent
  • Quick as they develop in five days
  • 3rd the price of mice
  • Easy to genetically manipulate
  • Develop exoutero
  • 200-300 eggs
29
Q

What is a morpholino?

A

Morpholinos are nucleotide analogs that recognize and bind short sequences (about 25 nucleotides) at the transcription start site or at splice sites of pre-mRNAs, and thus block the translation or proper splicing of the mRNA.

30
Q

Gene knockdown in zebrafish

A

Morpholinos block gene specific translation or splicing. They can be synthesised to target specific genes. This can cause the mutation in zerbafish.

31
Q

What is ENU?

A

It is N-ethyl-N-nitrosource. It is a potent mutagen that targets spermatogonial stem cells.
It causes point mutations at a rate of 1 per 700 gametes

32
Q

How are targeted mutations achieved?

A

Using CRISPR

33
Q

What is CRISPR/Cas9 system?

A

It is a bacterial adaptive immune system involving Cas9 (CRISPR associated protein) and
Clustered regularly interspaced short palindromic repeats (CRISPR). The protospacer adjacent motif guides RNA to bind to strands of genomic DNA. The Cas9 endonuclease binds to non-protospacer portion of gDNA + PAM of DNA. This causes double stranded breaks (DSB) 3bp upstream of PAM. The mutations can be introduced through NHEJ and HDR.

34
Q

What are protospacers?

A

Target sequence of guide RNA

35
Q

What are examples of double stranded breaks?

A
  • Non-homologous end joining: error prone, lots of additional nucleotides or deletes
  • Homologous directed repair
36
Q

How are mutation pathogenes proven to exist?

A

The gene knockdown can be rescued (reveresed) which will prove there is a variant causing the disease.

37
Q

Example of RNA rescue experiments

A

Autosomal recessive cerebellar ataxias (ARCAs) is a neurodegenerative disorders. They identified a mutation in CHP1 in patients by doing WES. They found that when injected RNA with chp1. It rescues the morpholiene.

Genomics (19 decks)

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