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Advanced molecules, membranes and cells > Genetic Manipulation 6 > Flashcards

Genetic Manipulation 6 Flashcards

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1
Q

Dolly the sheep paper

A

Wilmut et al 1997 - Nature

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2
Q

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

A

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

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3
Q

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

A

put nucleus into an enucleated occyte

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4
Q

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

A

direct injection or electrofusion

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5
Q

How is the oocyte with donor nucleus activated?

A

electrical or chemical activation

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6
Q

How does activation of the occyte work?

A

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

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7
Q

What is done with the oocyte after activation?

A

transfer to uterus of pseudopregnant female

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8
Q

List some uses of cloning

A

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

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9
Q

briefly describe how to make a transgenic farm animal

A

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

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10
Q

example of therapeutic transgenic farm animal

A

human factor 9 transgenic sheep - Schnicke 1997

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11
Q

What year was dolly the sheep cloned?

A

1996

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12
Q

What year was Polly the transgenic sheep cloned?

A

1997

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13
Q

Problems with cloned animals

A 
lung disease (Dolly)
viability of cloned embryos low
large offspring syndrome 
resp and circulatory problems
weak immune system 
premature ageing
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14
Q

perceived problems with cloned animals

A

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

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15
Q

Explain cloning which makes use of ES cells

A

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

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16
Q

paper detailing generation of dopaminergic neurons from ES cells

A

Cho et al 2008

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17
Q

technical challenges of stem cell technology

A

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

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18
Q

Paper - induction of pluripotent stem cells from fibroblasts

A

takahashi and yamanaka 2006

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

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19
Q

Transcription factors expressed to make IPS

A

c-Myc, Oct4, sox2, Klf4

20
Q

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

A

Oct4, nanog and sox2

21
Q

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

A

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

22
Q

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

A

retroviruses

23
Q

What is the Fbx15 gene?

A

expressed in pluripotent cells but not required for pluripotency

24
Q

What was Fbx15 replaced with?

A

neomycin resistance

25
Q

What was the point of adding in neomycin resistance?

A

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

26
Q

How many genes were required for IPS?

A

10 (IPS-MEF10 cells)

eventually 4 was enough

27
Q

Was nanog essential or dispensable for IPS?

A

dispensable

28
Q

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

A

teratoma formation

29
Q

what is a nude mouse?

A

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

30
Q

What happened to IPS-MEF3 cells in grafts?

A

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

31
Q

teratoma

A

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

32
Q

IPS-MEF10/4 - embryoid bodies

A

formed these like ES cells in culture

33
Q

embryoid bodies

A

clumps of differentiating cells

34
Q

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

A

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

35
Q

3 Key assays for ES cell like activity

A

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

36
Q

Roughly how does IPS work?

A

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

37
Q

Inject IPS cells into tetraploid mouse blastocyst - what happens?

A

make adult mice derived entirely from IPs cells

38
Q

What do tetraploid cells contribute to?

A

placenta but not embryo proper

39
Q

characteristics of tetraploid IPS mice

A

fertile, full ES like pluripotency of ES cells

40
Q

paper - “cure sickle cell” IPS

A

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

41
Q

Dopaminergic neurons - IPS

A

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

42
Q

Advantage of IPS cells over ES cells

A

just as good but avoid ethical issues

43
Q

worry about IPS cells

A

would form tumours

44
Q

haemangioblasts: IPS vs Es?

A

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

45
Q

Epigenetic memory

A

heterochromatin and stable methylation patterns

46
Q

Epigenetic memory - IPS cells

A

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

47
Q

Nuclear reprogramming

A

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

Advanced molecules, membranes and cells (20 decks)

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