disease models

Question 1

interfering RNA

Answer 1

can be used to down grade protein production by preventing RNA from being converted to proteins by the ribosome

Question 2

outline how CRISPR is used to knock- out/ knock in genes

Answer 2

1) sgRNA which has an identical sequence to target gene produced
2) added to cell and binds to target gene
3) secondary structure recruits Cas9
4) Cas 9 cleaves DNA leaving a ds cut

• -> added into a plasmid
• mammalian repair

Question 3

mammalian repair via NHEJ

Answer 3

deletion/ insertion - change in reading frame- mutates gene

Question 4

homologous end joining

Answer 4

adds in good form of gene

Question 5

how to make a mouse

Answer 5

1) order modified ES Cells (embryonic stem cells)
2) microinject into fertilised oocyte at blastocyst stage
3) transfer to pseudopregnant female
4) birth of litter
5) breed of chimeric offspring with wild type mice
6) screen for gremlin transmission and breed together
7) expansion

Question 6

natural disease models rarely exist because

Answer 6

normally diseased models do not survive

Question 7

difference between cells in a dish and in a body

Answer 7

• 2d vs 3d
• cell/cell contacts
• matrix rigidity
• 02 conditions

Question 8

knock-outs

Answer 8

performed to determine the physiological function of a gene

Question 9

knock-ins

Answer 9

performed to introduce specific mutations

Question 10

CRISPR-Cas9 method of producing model organisms

Answer 10

1) produce sgRNA
2) inject into fertilised one-celled oocyte
3) transfer to pseudopregnant females
4) skip chimeric stage
5) gremlin transmission occurs in all offspring
6) expansion

Question 11

benefits of using CRISPR-Cas9 method over embryonic stem cell in producing disease models

Answer 11

skips chimeric stage

- don’t need to beed more generations

Question 12

although effects of mutations in mice may be slightly different to the effects they exert on humans

Answer 12

they can provide a robust guide to the function of genes in mammalian species

Question 13

benefits of mutant mice

Answer 13

• can control environment-
• have similar genetic background
• quick to breed
• ceap to house
• short life spans
• similar genes to humans

Question 14

limitations of using mutant mice

Answer 14

• what strain
• what time of day
• carried out by male or female
• is cage clean
• mice not human

Question 15

all models are bad models

Answer 15

but some are useful

Question 16

example of when mice have been a bad disease model

Answer 16

when trying to replicate CF in mice

Question 17

why couldn’t CF be replicated in mice

Answer 17

although Cl- channels did close, ATPase channels not upregulated
= therefore immune clearance

Question 18

CF in humans and pigs

Answer 18

ATPase upregulated- conditions inhibit immune clearance

- as well as cl- channels being closed

Question 19

SCID mouse used for

Answer 19

tissue grafting

Question 20

target sequence for CRISPR CAS9

Answer 20

NGG- cut knock out gene

- think of it as being double stranded

Question 21

target sequence of sgRNA should be no longer than

Answer 21

20bp - therefore some genes may not be targetable

Question 22

NHEJ

Answer 22

silencing genes

Question 23

homologous directed repair

Answer 23

adds good exogenous genes- e.g. targeted for CF

Question 24

strategy to see if CRISPR stratify has deleted a region of a gene

Answer 24

design a primer specific to that gene

• try and PCR
• NGS

Question 25

NGS

Answer 25

is more expensive
- but you can quantitatively assess genome edits in your target sequence and other regions of the genome

–> good option if you have a large number of sample

Question 26

when seeing if you have transformed a cell

Answer 26

importnat to keep a set of control cells, for after sequencing comparisons