Genetic Manipulation 6 Flashcards
Dolly the sheep paper
Wilmut et al 1997 - Nature
What cell can be taken from a donor to be cloned?
gonadal cell, fibroblast, ES cells - every cell contains a complete genome
once you have a donor cell what is done with it?
put nucleus into an enucleated occyte
How is the nucleus of a donor cell put into an enucleated oocyte?
direct injection or electrofusion
How is the oocyte with donor nucleus activated?
electrical or chemical activation
How does activation of the occyte work?
donor DNA condenses, is reprogramed by cytoplasm of host and becomes totipotent again - loss of epigenetic silencing of genes
What is done with the oocyte after activation?
transfer to uterus of pseudopregnant female
List some uses of cloning
clone people, transgenic farm animals, study biology, clone pets/endangered animals, therapeutic cloning eg organs
briefly describe how to make a transgenic farm animal
zap DNA into cultured cells eg sheep fibroblast - do nuclear transfer into oocyte
example of therapeutic transgenic farm animal
human factor 9 transgenic sheep - Schnicke 1997
What year was dolly the sheep cloned?
1996
What year was Polly the transgenic sheep cloned?
1997
Problems with cloned animals
lung disease (Dolly) viability of cloned embryos low large offspring syndrome resp and circulatory problems weak immune system premature ageing
perceived problems with cloned animals
failure of epigentic reprogramming
accumulation of damage macromolecules
shortened telomeres - sheep and goats
retention of mutations during life of donor
Explain cloning which makes use of ES cells
take skin sample, inject nucleus into host oocyte - harvest ICM, make ES cells and differentiate them
paper detailing generation of dopaminergic neurons from ES cells
Cho et al 2008
technical challenges of stem cell technology
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
Paper - induction of pluripotent stem cells from fibroblasts
takahashi and yamanaka 2006
adult somatic cells IPS by forced expression of a few key transription factors
Transcription factors expressed to make IPS
c-Myc, Oct4, sox2, Klf4
What 3 transcription factors were known to maintain pluripotency in ES cells?
Oct4, nanog and sox2
What genes commonly expressed in tumours are required for long term maintenance of ES cell phenotype and rapid proliferation in culture?
Stat3, E-Ras, c-Myc, Klf4, B catenin
The 24 genes used to return somatic cells to ES like state were cloned into what?
retroviruses
What is the Fbx15 gene?
expressed in pluripotent cells but not required for pluripotency
What was Fbx15 replaced with?
neomycin resistance
What was the point of adding in neomycin resistance?
add neomycin to culture - only survivors should be cells in which IPS
How many genes were required for IPS?
10 (IPS-MEF10 cells)
eventually 4 was enough
Was nanog essential or dispensable for IPS?
dispensable
When IPS-MEF4 cells were injected into nude mice what happened?
teratoma formation
what is a nude mouse?
no thymus, no cell mediated immunity, do not reject grafts
What happened to IPS-MEF3 cells in grafts?
survived but formed tumours consisting of differentiated cells only - not pluripotent
teratoma
stem cell tumours consisting of differentiated cells from all 3 embryonic layers
IPS-MEF10/4 - embryoid bodies
formed these like ES cells in culture
embryoid bodies
clumps of differentiating cells
what happened when IPS-MEF 10 or 4 were injected into ICM?
form chimeric mice with mixture of host cells and IPS derive cells
3 Key assays for ES cell like activity
form embryoid bodies in culture
form chimeric mice when injected into ICM
form teratoma when injected into nude mouse
Roughly how does IPS work?
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
Inject IPS cells into tetraploid mouse blastocyst - what happens?
make adult mice derived entirely from IPs cells
What do tetraploid cells contribute to?
placenta but not embryo proper
characteristics of tetraploid IPS mice
fertile, full ES like pluripotency of ES cells
paper - “cure sickle cell” IPS
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
Dopaminergic neurons - IPS
Swistowski 2010
IPS cells from human blood and skin fibroblasts - dopaminergic neurons
transplant into rat model of parkinsons - improved behavioural deficits in rats
Advantage of IPS cells over ES cells
just as good but avoid ethical issues
worry about IPS cells
would form tumours
haemangioblasts: IPS vs Es?
IPS less efficient, more apoptotic, limited growth and expansion capacity
Epigenetic memory
heterochromatin and stable methylation patterns
Epigenetic memory - IPS cells
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
Nuclear reprogramming
nucleus of somatic adult cells all DNA but some permanently silenced (epigenetic)
reverse silencing - back to pluripotency bu cloning/IPS technology
