Pluripotency and iPS cells

Question 1

What is understood by Waddington’s epigenetic landscape model?

Answer 1

Cells can be reprogrammed to become pluripotent, if in the correct environment and then can become another specialised cell

Question 2

What happens during somatic cell nuclear transfer

Answer 2

the nucleus of a somatic cell is transplanted into an oocyte and an electric pulse triggers development and division

Question 3

What is a heterokaryon

Answer 3

during somatic cell nuclear transfer when the cell contains both nuclei and a mixed cytoplasm

Question 4

During somatic cell nuclear transfer, which nucleus is dominant

Answer 4

the nucleu of the pluripotent stem cell, NOT the somatic nucleus

Question 5

What are the mechanisms of maintenance of pluripotency in culture?

Answer 5

Growth factor/ cytokine signalling
Pluripotency genes and transcriptional network s

Question 6

What do culture conditions determine?

Answer 6

Cell signalling, which determines gene expression and so determines cell potency and determination

Question 7

What does the dominant nucleus do on a molecular level during somatic cell nuclear transfe r

Answer 7

reprogrammes the cell, through the expression of certain genes such as oct4. ES-like colonies are formed.

Question 8

What does Paracrine LIF signalling regulate? How?

Answer 8

Regulates ES cells specific gene expression by a activating the transcription factor STAT3
LIF binds to receptor - allows for phosphorylation of STAT3

Question 9

What are the 4 yamanaka genes and what do they do?

Answer 9

oct4, sox2, klf4, cMyc
they are the genes capable of reprogramming in human iPS cells

Question 10

Why is serum important in pluripotency?

Answer 10

It contains additional inhibitory signals of ES cells specific gene differentiation to neuroectoderm

Question 11

What gene important for pluripotency is a direct target of TGFb signalling?

Answer 11

Nanog

Question 12

How do reprogramming factors reconfigure the genome?

Answer 12

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

Question 13

What does BMP4 and LIF do in serum free-culture?

Answer 13

Blocks ES cell differentiation

Question 14

What happens to histones and DNA when sox2 and oct4 bind

Answer 14

activation and his tone acetylation

Question 15

What happens when oct4 alone binds DNA

Answer 15

repression of genes. his tone deacetylation and DNA methylation

Question 16

What is BIO?

Answer 16

A GSK3beta antagonist identified in a drug screen for molecules that promote pluripotency- led to focus on wnt pathway

Question 17

What happens when cMYC binds DNA

Answer 17

modulation of cell proliferation and metabolic genes

Question 18

How and why are drugs capable of altering reprogramming in SCNT?

Answer 18

because reprogramming TFs carry enzymatic activity so can be modulated by drugs to alter the impact on chromatin

Question 19

What is the role of wnt signalling to GSK3beta?

Answer 19

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

Question 20

How can cell reprogramming be used in a clinical setting

Answer 20

to source a diseased patients cells and conserve their genetics to observe the disease cells

Question 21

What is Oct4?

Answer 21

• BI-partite DNA binding POU domain transcription factor
• Transactivator and repressor
  -Oct4 mutant embryos die as blastocysts, ICMs fail to develop

Question 22

How can fibroblasts be reprogrammed?

Answer 22

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

Question 23

What happens to Oct4 mutant embryos?

Answer 23

They are defective in ICM formation

Question 24

What is Nanog?

Answer 24

A homeobox contains TF
Expression in pluripotent cells
Identified by its ability in a screen of gene overexpression to maintain ES cell pluripotency

Question 25

What occurs during Nanog mutant embryos?

Answer 25

They fail to develop epiblast

Question 26

List some prospects of iPS cell technology

Answer 26

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

Question 27

What genes have co-occupancy of gene promoters?

Answer 27

Oct4, Nanog, Sox2 coordinate transcriptional states

Question 28

What is genetic engineering?

Answer 28

introducing new genes into cells or editing genes that are already present

Question 29

What is a transgene

Answer 29

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

Question 30

What are chromosomal position effects? in terms of transgenes

Answer 30

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

Question 31

How can transgenes being subject to gene silencing be overcome

Answer 31

precisely target the endogenous genes in their own loci and captures all its regulatory sequences in its native state

Question 32

What is homologous recombination

Answer 32

strand exchanged from the endogenous genes in their with the new targeting vector. mediated by DNA repair machinery

Question 33

What is gene knocking in?

Answer 33

introducing new information to an exon

Question 34

How can you detect homologous recombination events?

Answer 34

using markers and GFP
distance between the markers changing shows there has been a polymorphism and the DNA has changed

Question 35

How can antibiotic resistance genes be used to monitor the uptake of transgenes s

Answer 35

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

Question 36

What is the role of the cre recombinase enzyme

Answer 36

identifies two adjacent loxP sites and deletes the dna between the sites

Question 37

How can cre activity be temporally regulated

Answer 37

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

Question 38

how can cre/lox be used as. molecular switches?

Answer 38

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

Question 39

What are genetically encoded biosensors used for

Answer 39

used as they show a flash of colour to report changes in intracellular levels such as Ca2+ influx

Question 40

How can CRISPR Cas9 be used in homologous recombination

Answer 40

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.