Lecture 21 - Gene Manipulation Strategies Flashcards
What does crispr do?
Defence mechanism from bacteriophage
What does crispr involve?
1) Cas9 endonuclease and a guide RNA (gRNA) cleave both DNA strands.
2) The gRNA should contain specific 20nt protospacer (target sequence) and designed upstream of a protospacer adjacent motif
What does crispr 9 recognise and what does this do?
Recognises PAM site and makes double stranded break 3 nucleotides upstream of PAM
What are the 3 ways crispr can introduce genomic modifications?
1) repaired by NHEJ - gene knock outs
2) repaired by HDR: use short Oligocene or DNA vectors with short homologous arms that flank the cleavage site
- introduce specific mutations, reporter genes (GFP etc) or loxP sites etc.
3) two gRNA target sites can be chosen - delete large genomic regions
Is crispr 9 officiant?
Yes all cells which undergo this will express the mutants
How do you generate mice with several mutations using crispr?
Inject cas9 and gRNA’s into pronucleu. Repair in Vigo using HDR (allows rapid generation of transgenic animals carrying specific modifications_
What do you do to stop off target effects as cas9 and gRNA is not absolutely specific?
Choose target sequences within genes with minimal homologous to other genes or regulatory regions
Wha reduces off target effects?
Mutant cas9 (nickase).
How does mutant cas 9 work?
Cleaves only one DNA strand leaving an overhand when used with 2 gRNA’s specific for opposite DNA strands.
NHEJ or HDR methods are used to knockout or introduce reporters
would 2 close gRNA target sites be unlikely to be present elsewhere in the genome and therefore increase the chance of 2 nicks being near one another?
Yes the are unlikely to be elsewhere in the genome but no they would reduce the chance of nicks being near one another
Did they efficiently generate genetically modified mice using crispr 9?
Yes - they put GFP in the reporter for EGF to show where it was in the cell.
What is conditional modification of the gene?
Conditional deletion of te gene
Why are conditional mutations useful?
- avoid early lethality of animal to analyse role of gene
- to activate mutations
Conditional mutations - what is cell specific activation useful?
analyse role of gene in a defined cell (cell specific inactivation)
Conditional mutations - why is inducible inactivation done?
analyse the role of gene at certain development stage (inducible inactivate)
Conditional modification - why is cell fare experiments done?
- permanent take cels with reporter gene to determine fate (cell fate experiments)
How do you conditionally translate a mutations?
DNA recombination can be driven in-vivo by expressing Cre-recombinase from a transgene
loxP sites are previously inserted by gene targeting to flank a critical sequence and create a loop
Results in loss of sequence from genome
What can you use instead of cre recombinase and loxP?
Flp recombinase and frt recognition sites
How do you make an inducable deletor mouse?
- Cre-ERT2 constructs to generate deletor mice: Cre fused to a mutant estrogen ligand-binding receptor domain ERT2, which responds to synthetic analogue oestrogen called tamoxifen.
ERT2 keeps Cre in cytoplasm but upon addition of tamoxifen can move to the nucleus and cause recombination
Once cre is in the nucleus what happens?
It binds to LoxP ad causes deletion/recombinant
Epithelial self-renewal is what?
An example of inducible deletions
How did they work out how the epithelial self renews?
They made knock out mice and fluorescents the Lgr5 expression at the bottom of the crypt
What happens in a normal LGR5 cells
Lgr5 drives GFP, IRES and CRE-ERT2
R26R stops LACZ
What happens when LGR5 cells are loaded with tomoxifen?
This gets rid of stop codon and LACZ is translated
What are the Lgr5 cells in the small intestine?
Adult stem cells
Will Lgr5 cells still give off LACZ even if there is no Lgr5?
Yes because they become independent off this
LGR5-CreERT2 mouse can be used to delete what?
APC tumour supressor gee specifically in gut stem cells
What do Lgr5-EGFP and intestinal stem cells transformed after loss of APC persist and fuel?
Rapid formation of microadenomas
What happens if yo use Ah-Cre debtor targets non-stem cell?
The population fails to drive intestinal neoplasia
Can mutant cats 9 be used to activate or repress genes of interest?
Yes
Can dcas9 cleave DNA and lead to mutation?
No and therefore if bound to an activator = activation and if bound to a repress or = represses
How do you reprogramme a genome?
Somatic nuclear transfer
How do you do somatic nuclear transfers?
Remove chromosomes from fertilised egg and replace with the nucleus from the somatic cell. The membrane of the donor cells and recipient oocytes fuse causing activation of embryo development initiated by electrical pulses
What are the types of cloning?
Reproductive cloning - generating an animal which contains a copy of the genome of another animal
Therapeutic cloning - generating ES cells from a cloned embryo with intents ion to generate cell types in vitro for regenerative medicine
Why is somatic nuclear cloning so inefficient?
During normal development there is lots of steps that need to be done for survival - DNA demethylation, chromatin remodelling, zygotes gene activation and this is not done during cloning
There is abnormal epigenetic regulation
Epigenetics in reprogramming - inheritance is independent of what?
Primary DNA sequence
Epigenetics in reprogramming - what is this mediated by?
Heritable but reversible modification to DNA or some heritable change to chromatin
What is required for normal development?
A maternal and paternal pronucleus
What is parent of origin imprinting?
Some gene are expressed or repressed depending on their parental origin resulting in monoallelic uniparental expression
Parents of origin imprinting - Where does imprinting occur?
Gametogenesis - some during male and some during female gametogenesis
Where do parent of origin imprinting persists through?
To zygote into somatic cell of adult animal
Can imprints be erased and re-established due to gender of embryo?
Yes
In primaordial germ cells are all m prints erased before being tuned on or off during female of male gametes?
Yes
Is there maintainance of imprints?
Yes
Where re I ruined genes found in chromosomes?
Clustered in certain regions
How do you show imprinted genes? Males
Mutate and turn of the gene that was meant to be expressed and on the one that wasn’t meant to be expressed and mate with a wild type - leaves an abnormal phenotype
How would you reveal an imprinted gene in a female y mutations?
Mutate and turn off gene that was meant to be on and turn on gene that wasn’t meant to be on and mate with wild type male. This leads to no phenotype
What are the effects of imprinted genes?
Feral growth, placenta growth and postnatal behavious
What are the effects of paternally expressed imprinted genes and maternally expressed genes on fetal growth?
Paternal infancies growth maternal suppresses growth
What do defects in imprinted genes lead to?
Disease and cancer
What is found close to or within imprinted genes?
Differentials methylated regions (DMR’s)
What are some other epigenetic modifications?
His tone acetylation and regulation of non-coding RNA is also implicated
Sometimes cloning can lead to normal eggs which means there must be reprogramming of differentiated cells into pluripotent ones. What could these be?
OCT4, SOX2, KLF4, c-MYC are sufficient to generate Induced Pluriotent stem cells (IPS) from mouse fibroblasts
Can you reprogramme differentiated cells (IPs) into pluripotent cells?
Yes
What are some potential therapeutic uses of IPS?
Transplant,
What are problems of potential therapeutic use of IPS cells?
Any cells undifferentiated could make tumours.
Cell types differentiated in culture may not be fully functional
Integration of cells and establishment proper contacts with other cells in tissues may be problematic.
