ESCs and transgenesis Flashcards
What are embryonic stem cells?
Pluripotent stem cells derived from the inner cell mass of the early mammalian embryo
What is the basics of what you can use ESC for?
Can be grown into culture, genetically modified and inserted into a blastocyst to develop a transgenic animal
What is the name of the cancer with many tissues?
Teratoma
Where do teratomas arise?
In the gonads
When were embryonic carcinoma cells first established?
In 1970s
On what were EC grown before?
On feeder cells
What happens when you don’t grow EC on feeder cells?
Produces mixed populations of ECs and more differentiated feeder-like cells
When was the first embryonic stem cell line established?
In 1981
What happened on 1984 after the stem cell line was established?
Embryonic stem cells gave rise to chimeric mice
Where do you isolate embryonic stem cells from?
Blastocysts
What do ES cells need apart from feeder cells?
Calf serum
What are BRL cells and what’s their problem?
Buffalo-rat liver cells, they secrete a protein that stops differentiation of ES cells, this is how it mantains ES cells
What was discovered in 1998?
Leukemia inhibiting factor
What is LIF?
LIF is a cytokine secreted by mice embryonic fibroblasts or BRL cells
What is the pathway that is activated by LIF and why is it important?
LIF activates the JAK/STAT signaling pathway that leads to the activation of transcription factor STAT3 which induces self-renewal genes
What happens if you grow ES cells with only LIF but no feeders?
ES show more differentiation, meaning that feeders have more factors important for pluripotency
What else do ES cells rely on and can be replaced by BMP signaling?
Serum
What type of signaling do cells also rely on?
BMP signaling
What can replace exogenous BMP signaling?
GSK and ERK inhibition
What is currently the best ES cell medium that keeps mouse ES in a ground state?
LIF+ERK and GSK inhibitors
What are the three stem pluripotency states ?
- ES cells in serum + LIF are in naive state
- ES cells in 2i + LIF are in ground state
EpiSCs are in primed state
What is BMP?
Bone morphogenic protein
What does BMP activate and how does it work?
it activates Id proteins which in turn inhibit differentiation, only works with LIF
what is a pathway that BMP inhibits that leads to pluripotency?
Erk-mitogen activated proteins
What is the function of Wnt signaling?
Differentiation of ES cells
WHat is GSK Why is it important?
A kinase on the Wnt signaling cascade, by inhibiting you inhibit differentiation
What is a 2i medium?
2 inhibitors (GSK and ERK) + LIF
What is the best serum free medium?
2i medium
What are EpiSC cells?
Another source of mouse pluripotent stem cells from E6.5 embryos (egg cylinder) that are pluripotent but cant generate germ cells
What major event happened in 1998?
The first ES cells derived from human blastocysts
What resembles human embryonic cells?
EpiSC in primed state
What is the pathway human ES cells rely on for pluripotency?
Activin/FGF signaling +2i
What are the core transcription factors for human embryonic stem cells?
Nanog, Sox2 and Oct3/4
What is the function of the transcription factors?
They regulate the pluripotency network that promotes self-renewal and restricts differentiation
What can substitute LIF/StAT3 signaling in mouse ES?
Nanog expression
What happens with Nanog KO ES cells
They rapidly lose pluripotency
What is a trophoblast state and what can lead to it?
It is placental cell type and Oct4 loss can lead to it
What happens when Oct4 or Sox2 is overexpressed? What is the conssequence?
Leads to loss of pluripotency , pluripotency factors need to be dosed just right
What is the relation between Sox2 and Oct4?
Sox2 boosts activity of Oct4
How can you rescue Sox2 defiency?
By forced overexpression of Oct3/4
What are the three steps of NHEJ?
- Cleaning ends, 2. Base removal, 3. End-end joining
How can you trick cells into homologous recombination?
With a donor template
When was the first transgenic mouse made?
In 1974
Give examples of 4 complex mouse models from genetic engineering
Knockout alleles, knockin alleles, conditional alleles, conditional activatable alleles using tetracyclin inducible promoter
How do conditional alleles get in?
Crossing Cre-mouse with LoxP mouse, cre comes with a tissue-specific promoterw
What mechanism is involved with gene activation by tetracyclin?
Tet-on system
