Genetic Manipulation 6

Question 1

Dolly the sheep paper

Answer 1

Wilmut et al 1997 - Nature

Question 2

What cell can be taken from a donor to be cloned?

Answer 2

gonadal cell, fibroblast, ES cells - every cell contains a complete genome

Question 3

once you have a donor cell what is done with it?

Answer 3

put nucleus into an enucleated occyte

Question 4

How is the nucleus of a donor cell put into an enucleated oocyte?

Answer 4

direct injection or electrofusion

Question 5

How is the oocyte with donor nucleus activated?

Answer 5

electrical or chemical activation

Question 6

How does activation of the occyte work?

Answer 6

donor DNA condenses, is reprogramed by cytoplasm of host and becomes totipotent again - loss of epigenetic silencing of genes

Question 7

What is done with the oocyte after activation?

Answer 7

transfer to uterus of pseudopregnant female

Question 8

List some uses of cloning

Answer 8

clone people, transgenic farm animals, study biology, clone pets/endangered animals, therapeutic cloning eg organs

Question 9

briefly describe how to make a transgenic farm animal

Answer 9

zap DNA into cultured cells eg sheep fibroblast - do nuclear transfer into oocyte

Question 10

example of therapeutic transgenic farm animal

Answer 10

human factor 9 transgenic sheep - Schnicke 1997

Question 11

What year was dolly the sheep cloned?

Answer 11

1996

Question 12

What year was Polly the transgenic sheep cloned?

Answer 12

1997

Question 13

Problems with cloned animals

Answer 13

lung disease (Dolly)
viability of cloned embryos low
large offspring syndrome 
resp and circulatory problems
weak immune system 
premature ageing

Question 14

perceived problems with cloned animals

Answer 14

failure of epigentic reprogramming
accumulation of damage macromolecules
shortened telomeres - sheep and goats
retention of mutations during life of donor

Question 15

Explain cloning which makes use of ES cells

Answer 15

take skin sample, inject nucleus into host oocyte - harvest ICM, make ES cells and differentiate them

Question 16

paper detailing generation of dopaminergic neurons from ES cells

Answer 16

Cho et al 2008

Question 17

technical challenges of stem cell technology

Answer 17

pathogen free culture facility
prevent spontaneous differentiation of ES cells
culture cells in defined and animal product free medium
minimise tumorigenic potential
overcome recipient immune response
homogenously produce cell types of need

Question 18

Paper - induction of pluripotent stem cells from fibroblasts

Answer 18

takahashi and yamanaka 2006

adult somatic cells IPS by forced expression of a few key transription factors

Question 19

Transcription factors expressed to make IPS

Answer 19

c-Myc, Oct4, sox2, Klf4

Question 20

What 3 transcription factors were known to maintain pluripotency in ES cells?

Answer 20

Oct4, nanog and sox2

Question 21

What genes commonly expressed in tumours are required for long term maintenance of ES cell phenotype and rapid proliferation in culture?

Answer 21

Stat3, E-Ras, c-Myc, Klf4, B catenin

Question 22

The 24 genes used to return somatic cells to ES like state were cloned into what?

Answer 22

retroviruses

Question 23

What is the Fbx15 gene?

Answer 23

expressed in pluripotent cells but not required for pluripotency

Question 24

What was Fbx15 replaced with?

Answer 24

neomycin resistance

Question 25

What was the point of adding in neomycin resistance?

Answer 25

add neomycin to culture - only survivors should be cells in which IPS

Question 26

How many genes were required for IPS?

Answer 26

10 (IPS-MEF10 cells) | eventually 4 was enough

Question 27

Was nanog essential or dispensable for IPS?

Answer 27

dispensable

Question 28

When IPS-MEF4 cells were injected into nude mice what happened?

Answer 28

teratoma formation

Question 29

what is a nude mouse?

Answer 29

no thymus, no cell mediated immunity, do not reject grafts

Question 30

What happened to IPS-MEF3 cells in grafts?

Answer 30

survived but formed tumours consisting of differentiated cells only - not pluripotent

Question 31

teratoma

Answer 31

stem cell tumours consisting of differentiated cells from all 3 embryonic layers

Question 32

IPS-MEF10/4 - embryoid bodies

Answer 32

formed these like ES cells in culture

Question 33

embryoid bodies

Answer 33

clumps of differentiating cells

Question 34

what happened when IPS-MEF 10 or 4 were injected into ICM?

Answer 34

form chimeric mice with mixture of host cells and IPS derive cells

Question 35

3 Key assays for ES cell like activity

Answer 35

form embryoid bodies in culture form chimeric mice when injected into ICM form teratoma when injected into nude mouse

Question 36

Roughly how does IPS work?

Answer 36

oct4 and sox2 = core transcription factors for maintaining pluripotency c-Myc = oncogene, lots of downstream targets, open DNA up for transcription Klf4 - represses p53, represses nanog

Question 37

Inject IPS cells into tetraploid mouse blastocyst - what happens?

Answer 37

make adult mice derived entirely from IPs cells

Question 38

What do tetraploid cells contribute to?

Answer 38

placenta but not embryo proper

Question 39

characteristics of tetraploid IPS mice

Answer 39

fertile, full ES like pluripotency of ES cells

Question 40

paper - "cure sickle cell" IPS

Answer 40

jacob et al 2007 human B-globin gene in mice mutated, IPS cells from tail fibroblasts, knock in B globin gen, differentiate and inject back in = cure

Question 41

Dopaminergic neurons - IPS

Answer 41

Swistowski 2010 IPS cells from human blood and skin fibroblasts - dopaminergic neurons transplant into rat model of parkinsons - improved behavioural deficits in rats

Question 42

Advantage of IPS cells over ES cells

Answer 42

just as good but avoid ethical issues

Question 43

worry about IPS cells

Answer 43

would form tumours

Question 44

haemangioblasts: IPS vs Es?

Answer 44

IPS less efficient, more apoptotic, limited growth and expansion capacity

Question 45

Epigenetic memory

Answer 45

heterochromatin and stable methylation patterns

Question 46

Epigenetic memory - IPS cells

Answer 46

Kim et al 2010 - retain some of methylation pattern of somatic cells, more prone to differentiate back into same sorts of cells not intrinsically as good as ES cells

Question 47

Nuclear reprogramming

Answer 47

nucleus of somatic adult cells all DNA but some permanently silenced (epigenetic) reverse silencing - back to pluripotency bu cloning/IPS technology