disease models Flashcards
interfering RNA
can be used to down grade protein production by preventing RNA from being converted to proteins by the ribosome
outline how CRISPR is used to knock- out/ knock in genes
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
mammalian repair via NHEJ
deletion/ insertion - change in reading frame- mutates gene
homologous end joining
adds in good form of gene
how to make a mouse
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
natural disease models rarely exist because
normally diseased models do not survive
difference between cells in a dish and in a body
- 2d vs 3d
- cell/cell contacts
- matrix rigidity
- 02 conditions
knock-outs
performed to determine the physiological function of a gene
knock-ins
performed to introduce specific mutations
CRISPR-Cas9 method of producing model organisms
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
benefits of using CRISPR-Cas9 method over embryonic stem cell in producing disease models
skips chimeric stage
- don’t need to beed more generations
although effects of mutations in mice may be slightly different to the effects they exert on humans
they can provide a robust guide to the function of genes in mammalian species
benefits of mutant mice
- can control environment-
- have similar genetic background
- quick to breed
- ceap to house
- short life spans
- similar genes to humans
limitations of using mutant mice
- what strain
- what time of day
- carried out by male or female
- is cage clean
- mice not human
all models are bad models
but some are useful
example of when mice have been a bad disease model
when trying to replicate CF in mice
why couldn’t CF be replicated in mice
although Cl- channels did close, ATPase channels not upregulated
= therefore immune clearance
CF in humans and pigs
ATPase upregulated- conditions inhibit immune clearance
- as well as cl- channels being closed
SCID mouse used for
tissue grafting
target sequence for CRISPR CAS9
NGG- cut knock out gene
- think of it as being double stranded
target sequence of sgRNA should be no longer than
20bp - therefore some genes may not be targetable
NHEJ
silencing genes
homologous directed repair
adds good exogenous genes- e.g. targeted for CF
strategy to see if CRISPR stratify has deleted a region of a gene
design a primer specific to that gene
- try and PCR
- NGS
NGS
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
when seeing if you have transformed a cell
importnat to keep a set of control cells, for after sequencing comparisons
