Pluripotency and iPS cells Flashcards
What is understood by Waddington’s epigenetic landscape model?
Cells can be reprogrammed to become pluripotent, if in the correct environment and then can become another specialised cell
What happens during somatic cell nuclear transfer
the nucleus of a somatic cell is transplanted into an oocyte and an electric pulse triggers development and division
What is a heterokaryon
during somatic cell nuclear transfer when the cell contains both nuclei and a mixed cytoplasm
During somatic cell nuclear transfer, which nucleus is dominant
the nucleu of the pluripotent stem cell, NOT the somatic nucleus
What are the mechanisms of maintenance of pluripotency in culture?
Growth factor/ cytokine signalling
Pluripotency genes and transcriptional network s
What do culture conditions determine?
Cell signalling, which determines gene expression and so determines cell potency and determination
What does the dominant nucleus do on a molecular level during somatic cell nuclear transfe r
reprogrammes the cell, through the expression of certain genes such as oct4. ES-like colonies are formed.
What does Paracrine LIF signalling regulate? How?
Regulates ES cells specific gene expression by a activating the transcription factor STAT3
LIF binds to receptor - allows for phosphorylation of STAT3
What are the 4 yamanaka genes and what do they do?
oct4, sox2, klf4, cMyc
they are the genes capable of reprogramming in human iPS cells
Why is serum important in pluripotency?
It contains additional inhibitory signals of ES cells specific gene differentiation to neuroectoderm
What gene important for pluripotency is a direct target of TGFb signalling?
Nanog
How do reprogramming factors reconfigure the genome?
DNA binding and epigenetic chromatin remodelling.
eg, oct4 turns on its own gene and activates other genes through gene networking.
find the targets of gene expression and recruit chromatin modifying proteins and bind DNA to either activate or repress at certain loci
What does BMP4 and LIF do in serum free-culture?
Blocks ES cell differentiation
What happens to histones and DNA when sox2 and oct4 bind
activation and his tone acetylation
What happens when oct4 alone binds DNA
repression of genes. his tone deacetylation and DNA methylation
What is BIO?
A GSK3beta antagonist identified in a drug screen for molecules that promote pluripotency- led to focus on wnt pathway
What happens when cMYC binds DNA
modulation of cell proliferation and metabolic genes
How and why are drugs capable of altering reprogramming in SCNT?
because reprogramming TFs carry enzymatic activity so can be modulated by drugs to alter the impact on chromatin
What is the role of wnt signalling to GSK3beta?
Modulates GSK3beta allowing nuclear accumulation of beta-date in and transcriptional regulation by collaboration with TCF factors
STAT3 transcription is a target for B-catenin, providing a point of cross-talk between wnt and LIF pathways
Wnt drives self renewal cell proliferation
How can cell reprogramming be used in a clinical setting
to source a diseased patients cells and conserve their genetics to observe the disease cells
What is Oct4?
- BI-partite DNA binding POU domain transcription factor
- Transactivator and repressor
-Oct4 mutant embryos die as blastocysts, ICMs fail to develop
How can fibroblasts be reprogrammed?
treat with drugs to enhance the expression of genes such as oct4, sox2, klf4 and cMyc to change them into functional cardiomyocytes with excitable potential
What happens to Oct4 mutant embryos?
They are defective in ICM formation
What is Nanog?
A homeobox contains TF
Expression in pluripotent cells
Identified by its ability in a screen of gene overexpression to maintain ES cell pluripotency
What occurs during Nanog mutant embryos?
They fail to develop epiblast
List some prospects of iPS cell technology
patient derived human disease models
disease mechanisms and drug screens
generation of autologous pluripotent stem cells
cell therapy without immune rejection
gene therapy coupled to cell therapy
What genes have co-occupancy of gene promoters?
Oct4, Nanog, Sox2 coordinate transcriptional states
What is genetic engineering?
introducing new genes into cells or editing genes that are already present
What is a transgene
introduced into a new organism to introduce a new gene with a new function
has a start and stop codon and enhancer sequence to control the expression of a gene in specific cell types at specific times
What are chromosomal position effects? in terms of transgenes
the transgene can integrate anywhere in the genome, despite the promoter and enhancer, the transgene can be influenced by the environment where it is integrated such as the presence of regulatory information on a neighbouring gene
How can transgenes being subject to gene silencing be overcome
precisely target the endogenous genes in their own loci and captures all its regulatory sequences in its native state
What is homologous recombination
strand exchanged from the endogenous genes in their with the new targeting vector. mediated by DNA repair machinery
What is gene knocking in?
introducing new information to an exon
How can you detect homologous recombination events?
using markers and GFP
distance between the markers changing shows there has been a polymorphism and the DNA has changed
How can antibiotic resistance genes be used to monitor the uptake of transgenes s
using genes with antibiotic resistance, kills the cells that does not uptake the DNA, taking up the DNA gives cells resistance to a drug meaning that they survive and express the gene
What is the role of the cre recombinase enzyme
identifies two adjacent loxP sites and deletes the dna between the sites
How can cre activity be temporally regulated
by taximofen which binds to an oestrogen receptor which indirectly modifies cre. allows the entry of cre into the nucleus to modify DNA to be regulated
how can cre/lox be used as. molecular switches?
by keeping a gene silent in development through putting a stop codon before the gene. when adding cre, stop signal can be removed to express the gene of interest
What are genetically encoded biosensors used for
used as they show a flash of colour to report changes in intracellular levels such as Ca2+ influx
How can CRISPR Cas9 be used in homologous recombination
can act as a flag for an incoming piece of DNA for homologous recombination. make dsDNA breaks to engage cells DNA repair mechanisms at the site of recombination integration.
