ESCs and transgenesis

Question 1

What are embryonic stem cells?

Answer 1

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

Question 2

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

Answer 2

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

Question 3

What is the name of the cancer with many tissues?

Answer 3

Teratoma

Question 4

Where do teratomas arise?

Answer 4

In the gonads

Question 5

When were embryonic carcinoma cells first established?

Answer 5

In 1970s

Question 6

On what were EC grown before?

Answer 6

On feeder cells

Question 7

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

Answer 7

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

Question 8

When was the first embryonic stem cell line established?

Answer 8

In 1981

Question 9

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

Answer 9

Embryonic stem cells gave rise to chimeric mice

Question 10

Where do you isolate embryonic stem cells from?

Answer 10

Blastocysts

Question 11

What do ES cells need apart from feeder cells?

Answer 11

Calf serum

Question 12

What are BRL cells and what’s their problem?

Answer 12

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

Question 13

What was discovered in 1998?

Answer 13

Leukemia inhibiting factor

Question 14

What is LIF?

Answer 14

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

Question 15

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

Answer 15

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

Question 16

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

Answer 16

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

Question 17

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

Answer 17

Serum

Question 18

What type of signaling do cells also rely on?

Answer 18

BMP signaling

Question 19

What can replace exogenous BMP signaling?

Answer 19

GSK and ERK inhibition

Question 20

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

Answer 20

LIF+ERK and GSK inhibitors

Question 21

What are the three stem pluripotency states ?

Answer 21

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

Question 22

What is BMP?

Answer 22

Bone morphogenic protein

Question 23

What does BMP activate and how does it work?

Answer 23

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

Question 24

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

Answer 24

Erk-mitogen activated proteins

Question 25

What is the function of Wnt signaling?

Answer 25

Differentiation of ES cells

Question 26

WHat is GSK Why is it important?

Answer 26

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

Question 27

What is a 2i medium?

Answer 27

2 inhibitors (GSK and ERK) + LIF

Question 28

What is the best serum free medium?

Answer 28

2i medium

Question 29

What are EpiSC cells?

Answer 29

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

Question 30

What major event happened in 1998?

Answer 30

The first ES cells derived from human blastocysts

Question 31

What resembles human embryonic cells?

Answer 31

EpiSC in primed state

Question 32

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

Answer 32

Activin/FGF signaling +2i

Question 33

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

Answer 33

Nanog, Sox2 and Oct3/4

Question 34

What is the function of the transcription factors?

Answer 34

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

Question 35

What can substitute LIF/StAT3 signaling in mouse ES?

Answer 35

Nanog expression

Question 36

What happens with Nanog KO ES cells

Answer 36

They rapidly lose pluripotency

Question 37

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

Answer 37

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

Question 38

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

Answer 38

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

Question 39

What is the relation between Sox2 and Oct4?

Answer 39

Sox2 boosts activity of Oct4

Question 40

How can you rescue Sox2 defiency?

Answer 40

By forced overexpression of Oct3/4

Question 41

What are the three steps of NHEJ?

Answer 41

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

Question 42

How can you trick cells into homologous recombination?

Answer 42

With a donor template

Question 43

When was the first transgenic mouse made?

Answer 43

In 1974

Question 44

Give examples of 4 complex mouse models from genetic engineering

Answer 44

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

Question 45

How do conditional alleles get in?

Answer 45

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

Question 46

What mechanism is involved with gene activation by tetracyclin?

Answer 46

Tet-on system