functional genome

Question 1

What is next generation sequencing (WES, WGS)?

Answer 1

• rapid modern method for highthroughput DNA sequencing
• whole exome sequencing (WES) used to capture sequence of the coding region of the genome.
• aim to identify potentially disease causing variant in medicine

Question 2

What is bioinformatics ?

Answer 2

1. WES data is subjected to a prioritisation filtering protocol.
2. 15-20,000 coding SNPs reduced to one or several candidate genes
3. checked for co-segregation (family members) and validated by sanger sequence.

Question 3

What are the key assumptions of bioinformatics?

Answer 3

• ignores structural variants and other forms of genetic variation
• assumes causal variant is coding , ignoring regulatory elements and other non- variants outside of exon definition.
• assumes causal variant alters protein sequence, ignoring rare cases of functional synonymous changes.
• assumes causal variant has complete penetrance
• assumes causal variant has complete detectance.

Question 4

Why does filtered WES variants not prove causality?

Answer 4

• despite all evidence towards a certain gene the correct gene could still be missed due to
  1. poor coverage of sequencing.
  2. mutation not in coding region (so may be in promoter or enhancer away from coding region)

so you can’t give diagnosis soley based on filtered WES variants.

Question 5

what is an example of a disease identified by whole exome sequencing using sample blood/tissue biopsies?

Answer 5

• congenital muscular dystrophy

- patients with this disease have a variant in the myosin light 1 (MYL1) gene.

Question 6

What does genetic variation in MYL1 variant cause in patients?

Answer 6

1. homozygous splice acceptor variant predicted to cause in frame skip of exon 5
2. missense mutation in exon 5, substitution of a highly conserved amino acids.
   => gene expressed in fast twitch muscle, patients have reduced fast muscle fibres.

Question 7

What technique is used to identify the variation in the tissue and blood samples?

Answer 7

• western blotting analysis reveals apparent loss of MYL1/and variants in patients.

Question 8

What is cell culture techniques (in vitro)?

Answer 8

• removal of cell from an animal and subsequent growth in favourable conditions.
• primary cells have finite divisions but can be immortalised to provide continuous source.
• provides a cheap, rapid and reproducible model for studying normal physiology and biochemistry
• good alternative to using animal models

Question 9

What is the method of RNAi mediated gene silencing?

Answer 9

• based on endogenous microRNA gene silencing
• modified to induce GOI complementary sequence
• packaged in a DNA plasmid, expression controlled by a RNA polymerase III promoter
• 50 -70 nucleotide, exit nucleus, cleaved by nuclease called DICER in cytoplasm.
• cleaved segment 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.

Question 10

what is an example of ShRNA?

Answer 10

=> PDZRN3

• was found to be developmentally regulated in skeletal muscle
• C212 cells, immortalised mouse myoblasts
• PDZRN3 siRNA blocks upregulation of PDZRN3 and MHC
• Knock down of PDZRN3 : inhibition of myotube formation and MHC expression

Question 11

What methods can we use to identify where gene of interest (GOI’s) encoded protein is localised?

Answer 11

1. antibody staining : protein of interest , downstream target
2. transfect cells with GFP tagger GOI (CMV promoter)
3. transfect cells with GFP tagged mutated GOI (CMV promoter.)

Question 12

What is an example of a disease where you lose muscle?

Answer 12

• Deuche muscular dystrophy
• mutation in x chromosome
• could be bc exon 44 is deleted leading to premature stop codon
• exon 45 skipping
• frame shift
• exon 44 knock in.

Question 13

Why is cell culture not enough?

Answer 13

• cells behave differently in a petri dish compared to how they behave in a whole organism.
• does not stimulate acc conditions, lacks tissue signalling.
• no info on gene expression and function with regards to developmental phenotype.

Question 14

How do we make a knock out model in mouse?

Answer 14

=> construct a targetting vector and introduce to nucleus of pluripotent ES cells.
=> homologous regions integrates in the cassette. Embryonic Stem cells selected based on ABr.
=> +ve embryonic stem cells grown to blastocytes and implanted into pseudopregnent recipient mice.

Question 15

What is cre- lox system?

Answer 15

• involves the use of cre recombinase enzyme and recombinase recognition sites referred to as loxP sites.
• loxP sites are 32 base pairs consensus sequences which have 8 base cores in 2 inverted repeats
• ## these inverted repeats give loxP sites their capacity for directionality

Question 16

Why are zebra fish so good to study?

Answer 16

• eggs are optically transparent
• they develop ex utereo, outside the mothers body
  which allows us to follow the development of the embryo just under a microscope which isnt the case for mice.
• embryo develop very quickly (24hrs)
• female produces lots of egg 200 x per lay which helps study many embryo at once(representative)
• aquatic environment so drugs can be added which is vital in drug discovery
• zebra fish are small and contain fewer cells so they are easy to trace and develop

Question 17

What are the stages of development of zebra fish?

Answer 17

• 24 hrs : embryo develops
  = embryo has completed somitogenesis and already reasmbles an adult fish.
  5 days : UI brain, trunk, myotome muscle develops
• they are able to hunt for their own prey

Question 18

how do we make zebrafish gene knockdown?

Answer 18

=> antisense morpholino technology
- MO are synthetic oligonucleotides and compose of 25 nucleotide chains (similar to DNA/RNA) but rather than having ribose backbone they have morpholine backbone which makes them incredibly stable which means they dont stick to random places in the cell which might give them advantage of being less toxic

Question 19

What are 2 ways morpholinos work?

Answer 19

1. block genes specific translation by targetting the initiation site on the messenger RNA so ribosomal blocks the ribosomal machinery from translating the mRNA into protein.
2. blocking splicing :
   morphilinos can be designed against slice and site (against splice donor site) blocks the spliceosome from doing its job and then we get intronic inclusion rather than intron being spliced out.

Question 20

What are shRNA?

Answer 20

Artificial RNA with target hairpin turn that can be used to silence target gene expression via RNA interference
Used for gene knockdown: experimental technique by which the expression of one or more gene is reduced in an organism.
Used in in vitro experiments.