Lecture 10: induction of pluripotency and a cell's identity Flashcards
(23 cards)
transcription factors can be thought of a s
- defining a cell type
- SOX2, NANAG, OCT4 + others define an ES cell
metaphor for cellular differentiation:
- Waddington
- differentiation is like a ball rolling down a hill
- Valleys = epigenetic restriction
- cells can’t just change easily form one ‘state’ to another
- can change state with correct factors
two ways or resetting the cell state:
Cloning
Inducing pluripotency
Resetting the cell state Cloning:
- a cell nucleus is manipulated to ‘reset’ its state. What has to be reset?
- Gene expression: somatic genes turned off, embryonic genes turned on
- Methylation: reset back to a ‘totipotent’ configuration
- chromatin: Remodelled
Resetting the cell state: Inducing Pluripotency
- there must be factors in oocytes that are resetting the state
- a set of sequential chromatin & gene expression changes
You are ____ factors to drive iPS formation
OVEREXPRESSING
- how do u turn the expression off?
- lintivirus will integrate but then be silenced
- Alternative: use a non-integrating method
non-integrating delivery of reprogramming factors: 3 types
- Vector-based approach
- Protein based approaches
- Chemicals based reprogramming
Vector-based approach
- transient transfection
- adenovirus
- floxed vectors
- transposon
- mRNA
protein based approaches:
TAT-fusion
chemicals used reprogramming:
VPA, 5-AzaC
delivery of reprogramming factors RETROVIRUS delivery
can only transduce DIVIDING cells
delivery of reprogramming delivery: LENTIVIRUS delivery
can transduce both DIVIDING and NON-DIVIDING cells
in reprogramming factors using virus’ what does Cre do
recombinase recognises LOX sites. Excises the intervening sequences BUT leaves ONE lox site behind
a Transposable element (TE or transposon) =
- DNA sequence that can change its position within a genome
- can create and remove mutations
Transposase:
enzyme that binds to the end of a transposon: catalyses the movement of the transposon to another part of the genome..
by:
- cut and paste
- a replicative transposition
Delivery of reprogramming factors: Episomal:
-Episomal replication: some vectors can replicate EXTRA_CHROMOSOMAL i.e remains outside the chromosomes. If conc limiting can be diluted by cell division
delivery of reprogramming factors: mRNA
Exogenous mRNAs can trigger an ancient anti-viral response – which causes degredation. You need to use drugs to supress this degradation + 5’-protected mRNA
how do you identify bone fide iPS colonies?
- Morphology
- protein expression
- gene expression
- differentiation
- epigenetics
what other cells have iPS cells been derived from?
epidermis, neuron, blood, beta-call.
In reality, it appears any cell can be programmed to an iPS cells
iPS compared to ES
- Both pluripotent BUT iPS unlike ES carry the genotype of the parent cells.
- This means you have captured a particular genotype potentially forever (remember iPS cell can grow indefinitely).
- Therefore, you could potentially make iPS cells from different genotypes (including people with diseases)
what can we use induced pluripotent cells for?
- pluripotent stem cells grow forever
- compare diseased iPS cells with normal iPS cells & COMPARE
- is differentiation affected? (is the disease caused by the cells not forming properly)
- is the phenotype affected? (is it caused by the cells not functioning properly)
- DRUG SCREEN
what diseases fibroblasts have iPS cells been derived from?
-Duchene muscular dystrophy
-PArkinsons
-ADA-SCID
-type 1 diabetes
Gaucheries disease
-Lesch-Nyhan syndrome
transdifferentiation: Direct reprogramming
- can we change any cell into ant other cell???
- lots have been competed so far
- predict TF needed
- approx. 2000 TF
- approx 20 TF expressed in a cell type
- -could 3-5 TF work??
