The functional genome

Question 1

What are the two examples of next generation sequencing?

Answer 1

• Whole Exome Sequencing (WES) - used to capture the sequence of the coding region of the genome
• Whole Genome Sequencing (WGS) - captures the whole thinf (not always necessary)

Rapid, modern methods for high throughput DNA seq. They aim to identify potential disease causing genetic variants

Question 2

What are the steps involved in candidate gene filtering using WES?

Answer 2

• The first step of filtering is targeted sequencing of exons
• Then we remove synonymous variants
• After that we remove previously identified variants
• Finally we restrict to variants fitting dominant/recessive model of inheritance
• • This allows for 15 - 20,000 coding SNPs to be reduced to one or several candidate genes
    
    • Checked for co-segregation and validated by sanger sequencing

Question 3

How do we prove that a variant we have detected is pathogenic?

Answer 3

To prove that a variant is pathogenic we need further evidence

• Detection of Protein
  
  • -if no protein present, it is a good indication that that variant has a detrimental effect on protein
• Tissue/Cell Expression
  
  • -if examining a specific disorder of a tissue and gene is expressed in that tissue but not other tissue, then it is a good indication that the candidate gene is the correct one
• Knockdown/Overexpression Effects
  
  • -examine how the knockdown or overexpression of gene of interest changes the phenotype of the cell line
• Development of Cells/Tissues
  
  • -examine variants which might change the development of the tissue
• Molecular Mechanism of Action
  
  • -does the gene of interest have common pathways which are already known to be involved in the disease?

Question 4

How do you detect if a protein is affected from a genetic variant?

What is a disadvantage of this technique?

Answer 4

If gene is expressed in blood or tissue, take a biopsy and have a look to see if that protein is expressed in affected individual.

• However, gene of interest will not always be expressed in the blood and might not be in an accessible affected tissue, making this technique very difficult.
  
  • To counter this, we can start making in vitro cell culture models

Question 5

Describe cell culture techniques

List their advantages

Answer 5

Cells from animal removed and grown in favourable conditions in an artificial environment

primary cells (first taken out) have finite division but can be immortalised to provide continuous source

• cheap, rapid and reproducible model for studying normal physiology and biochemistry of cells
• reduces number of animals used in research; less restrictions

Question 6

What are the three techniques used to detect if a protein is affected by a genetic variant?

Answer 6

1. Gene Knockdown (micro-RNA gene silencing technique)
2. Protein Localisation
3. Induced pluripotent stem cells (IPSC)

Question 7

How is gene knockdown carried out?

Answer 7

RNAi mediated gene silencing is one way of knocking down gene function

1) Gene of interest is packaged in DNA plasmid with RNA polymerase II promoter which controls its expression
2) When transfected into nucleus, short hairpin RNA (ShRNA) is transcribed and the protein Exportin-5 exports it through the nucleopore into the cytoplasm
3) Dicer protein complex cleaves RNA. Cleaved segments then bind to RNA induced silencing complex (RISC) and there is direct cleavage and degradation of complementary mRNA.

Question 8

How does SiRNA differ to ShRNA?

Answer 8

similar to ShRNA, chemically synthesized, but not vector based

Question 9

How does protein locallisation work?

Answer 9

We can look at where the gene of interest’s encoded protein is localised:

• transfect cells with green fluorescent protein tagged gene of interest (CMV promoter)
• transfect cells with GFP tagged mutated gene of interest (CMV promoter)
• antibody staining of protein interest and downstream target

Question 10

How do induced pluripotent stem cells work?

Question 11

What is the process involved in induced pluripotent stem cell in cell culture?

Answer 11

Adult cells can be artificially induced to dedifferentiate and revert to pluripotent stem cells capable of becoming many types of cells e.g taking dermal cells, de differentiating and then re-differentiating into a muscle fibre

Useful in Duchenne Muscular Dystrophy, as patients have a mutation in gene dystrophin required for muscle fibre integrity (stop codon in exon 45 and missing exon 44).

TALEN or CRISPR genes can be used to cause changes in DNA which result in:

• exon skipping (translated DNA is in frame, but missing exon 44 and 45), causing a truncated but functional dystrophin protein
• frameshift mutations which produce a functioning dystrophin
• exon 44 knock in

Question 12

Why is a cell culture not enough to determine if a candidate gene variant is pathogenic?

Answer 12

• Cells behave differently in a petri dish than to how they behave in a whole organism
• Doesn’t stimulate the actual conditions inside an organism i.e signals from other tissues
  
  • Looking at cells in a dish doesnt provide information on gene expression and function with regards to developmental phenotypes
• This is why ANIMAL RESEARCH comes in

Question 13

What are the advantages of using a mouse as a mammalian model for human genetic disease?

Answer 13

• -Accelerated lifespan (1 year is 30 human years)
• -Small and reproduces quickly
• -Mammals therefore genetically similar to humans
• • More ethical than using larger animals

Question 14

How do you make a mutant mouse?

Answer 14

1) Take out embryonic stem cells
2) Introduce changes in DNA through homologous recombination and select for cells expressing desired gene
3) Embryonic stem cells injected into inner cell mass of blastocyst in mouse
4) Implanted into pseudopregnant female mouse
5) Test offspring for presence of gene

Question 15

What are the advantages of using a zebrafish as a vertebrate model for human genetic disease?

Answer 15

• Cheap
• Grow and breed easily (produce many eggs)
• Easy to genetically manipulate
• Develop fast

Question 16

What can we use in gene knockdown for zebrafish?

Answer 16

• Morpholino oligonucleotides (MOs) used to knock down gene function which has a morpholino backbone instead of a ribose backbone
  
  • MOs can be synthesised to target the start site of a gene blocking translation or targetting the splice site

Question 17

What do morpholinos do?

What is a problem with using morpholinos?

Answer 17

Used in gene knockdown of zebrafish, will MO’s block gene specific translation or splicing

Problem: They can have off-target genetic effects (toxic effects)

Question 18

What can we use to produce a mutant zebrafish?

Answer 18

Methods:

1) Bathing zebrafish in ENU (N-Ethyl-Nitrosourea)
2) CRISPR/Cas9 System

Question 19

What is an ENU and what does it cause?

Comment on reverse and forward genetics

Answer 19

1) BATHING FISH IN ENU

• Potent mutagen targeting spermatogonial stem cells
  
  • Causes point mutations at a rate of 1 per 700 gametes

Then you can cross the mutagenized males with females and look at their offspring you can do

1) Phenotypes based screening (forward genetics) - look for obvious changes e.g lack of eyes

2) Genotype based screening (reverse genetics) - taking DNA out of the fish and find mutations

Question 20

Describe the CRISPR-Cas 9 system

Answer 20

a technique for editing genes in living cells, involving a bacterial protein called Cas9 associated with a guide RNA complementary to a gene sequence of interest

Cas9 causes double stranded DNA breaks, and cell repairs this by non-homologous end joining or homologous directed repair, adding or deleting nucleotides, causing mutations

Question 21

What are RNA resuce experiments?

Answer 21

Provide proof of whether a variant is causing the disease is if you can resuce the genetic knockdown