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Epigenetics > ESCs and transgenesis > Flashcards

ESCs and transgenesis Flashcards

1
Q

What are embryonic stem cells?

A

Pluripotent stem cells derived from the inner cell mass of the early mammalian embryo

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2
Q

What is the basics of what you can use ESC for?

A

Can be grown into culture, genetically modified and inserted into a blastocyst to develop a transgenic animal

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3
Q

What is the name of the cancer with many tissues?

A

Teratoma

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4
Q

Where do teratomas arise?

A

In the gonads

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5
Q

When were embryonic carcinoma cells first established?

A

In 1970s

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6
Q

On what were EC grown before?

A

On feeder cells

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7
Q

What happens when you don’t grow EC on feeder cells?

A

Produces mixed populations of ECs and more differentiated feeder-like cells

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8
Q

When was the first embryonic stem cell line established?

A

In 1981

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9
Q

What happened on 1984 after the stem cell line was established?

A

Embryonic stem cells gave rise to chimeric mice

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10
Q

Where do you isolate embryonic stem cells from?

A

Blastocysts

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11
Q

What do ES cells need apart from feeder cells?

A

Calf serum

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12
Q

What are BRL cells and what’s their problem?

A

Buffalo-rat liver cells, they secrete a protein that stops differentiation of ES cells, this is how it mantains ES cells

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13
Q

What was discovered in 1998?

A

Leukemia inhibiting factor

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14
Q

What is LIF?

A

LIF is a cytokine secreted by mice embryonic fibroblasts or BRL cells

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15
Q

What is the pathway that is activated by LIF and why is it important?

A

LIF activates the JAK/STAT signaling pathway that leads to the activation of transcription factor STAT3 which induces self-renewal genes

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16
Q

What happens if you grow ES cells with only LIF but no feeders?

A

ES show more differentiation, meaning that feeders have more factors important for pluripotency

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17
Q

What else do ES cells rely on and can be replaced by BMP signaling?

A

Serum

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18
Q

What type of signaling do cells also rely on?

A

BMP signaling

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19
Q

What can replace exogenous BMP signaling?

A

GSK and ERK inhibition

20
Q

What is currently the best ES cell medium that keeps mouse ES in a ground state?

A

LIF+ERK and GSK inhibitors

21
Q

What are the three stem pluripotency states ?

A
  • ES cells in serum + LIF are in naive state
  • ES cells in 2i + LIF are in ground state
    EpiSCs are in primed state
22
Q

What is BMP?

A

Bone morphogenic protein

23
Q

What does BMP activate and how does it work?

A

it activates Id proteins which in turn inhibit differentiation, only works with LIF

24
Q

what is a pathway that BMP inhibits that leads to pluripotency?

A

Erk-mitogen activated proteins

25
Q

What is the function of Wnt signaling?

A

Differentiation of ES cells

26
Q

WHat is GSK Why is it important?

A

A kinase on the Wnt signaling cascade, by inhibiting you inhibit differentiation

27
Q

What is a 2i medium?

A

2 inhibitors (GSK and ERK) + LIF

28
Q

What is the best serum free medium?

A

2i medium

29
Q

What are EpiSC cells?

A

Another source of mouse pluripotent stem cells from E6.5 embryos (egg cylinder) that are pluripotent but cant generate germ cells

30
Q

What major event happened in 1998?

A

The first ES cells derived from human blastocysts

31
Q

What resembles human embryonic cells?

A

EpiSC in primed state

32
Q

What is the pathway human ES cells rely on for pluripotency?

A

Activin/FGF signaling +2i

33
Q

What are the core transcription factors for human embryonic stem cells?

A

Nanog, Sox2 and Oct3/4

34
Q

What is the function of the transcription factors?

A

They regulate the pluripotency network that promotes self-renewal and restricts differentiation

35
Q

What can substitute LIF/StAT3 signaling in mouse ES?

A

Nanog expression

36
Q

What happens with Nanog KO ES cells

A

They rapidly lose pluripotency

37
Q

What is a trophoblast state and what can lead to it?

A

It is placental cell type and Oct4 loss can lead to it

38
Q

What happens when Oct4 or Sox2 is overexpressed? What is the conssequence?

A

Leads to loss of pluripotency , pluripotency factors need to be dosed just right

39
Q

What is the relation between Sox2 and Oct4?

A

Sox2 boosts activity of Oct4

40
Q

How can you rescue Sox2 defiency?

A

By forced overexpression of Oct3/4

41
Q

What are the three steps of NHEJ?

A
  1. Cleaning ends, 2. Base removal, 3. End-end joining
42
Q

How can you trick cells into homologous recombination?

A

With a donor template

43
Q

When was the first transgenic mouse made?

A

In 1974

44
Q

Give examples of 4 complex mouse models from genetic engineering

A

Knockout alleles, knockin alleles, conditional alleles, conditional activatable alleles using tetracyclin inducible promoter

45
Q

How do conditional alleles get in?

A

Crossing Cre-mouse with LoxP mouse, cre comes with a tissue-specific promoterw

46
Q

What mechanism is involved with gene activation by tetracyclin?

A

Tet-on system

Epigenetics (9 decks)

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