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Title: Induced Pluripotent Stem Cells
Description: Useful short questions and answers I wrote based on UCL biomedical sciences second year Induced Pluripotent Stem Cells lecture. To get a 2.1/1st you really should be able to answer all of these.
Description: Useful short questions and answers I wrote based on UCL biomedical sciences second year Induced Pluripotent Stem Cells lecture. To get a 2.1/1st you really should be able to answer all of these.
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Induced Pluripotent Stem Cells
Questions
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What three principles contributed to the discovery of iPSCs
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Roughly how many protein coding genes are there? What are they split into?
Who first tried SCNT? What did he do?
How can it be proven that the somatic cell was fully differentiated?
Is the success rate very good?
Who cloned the first larger animal? What was the experiment?
What are the implications of SCNT for SCs?
Define pluripotency
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where do pluripotent cell lines come from?
How many genes in PSCs and how many are needed to induce PSCs? Name them
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What is gene transcription regulated by?
What is a heterokaryon?
Who performed the first human SCNT to form ESCs? Why did it fail in previous attempts?
What was the experiment?
What implications does this have?
What a repressive epigenetics?
What are activating?
What is the next step in experimentation?
What are the problems with iPSCs?
What cannot be used for iPSC for humans? Why?
Discuss disease modelling and cell therapy in iPSCs
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Who was the first to cure an animal model of a disease using iPSCs?
List the 6 advantages of iPSCs and 3 disadvantages
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Differentiated cells that have been reprogrammed back to a state of pluripotency and other
cell fates by treatment with defined factors
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Somatic cell nuclear transfer
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Transcription factors
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SCNT showed that the nucleus of differentiated cells retains the same genetic information as
early ESC, and that these genes can be reactivated as they are just silenced
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In differential gene loss, genes not required
by differentiated cells are permanently inactivated
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6-7% housekeeping and the rest are tissue selective so
undergo differential gene expression
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John Gurdon in 1962
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A cloned albino frog could be
formed, showing the genome could be reprogrammed
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Do experiment using a nucleus from a B cell producing antibodies (plasma cell), which is
definitely a terminally differentiated cell
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3%
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Campbell and Wilmut 1997 – Black faced sheep (BFS) enucleated egg
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Nucleus transferred into egg
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Got a
WFS clone
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Embryonic, pluripotent SCs can be obtained from that cloned embryo, which genetically
match the donor, with the potential to differentiate into patient specific cells
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Pluripotent cells from the inner cell mass can differentiate into all tissues from the three
germs layers, but not into extra-embryonic tissues
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Development of techniques which allowed cell culture of pluripotent SC lines
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It was found that enforced expression could switch mature cell
types into another
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This caused the
activation of human muscle specific genes in human liver cell
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He identified 3 genes specifically
transcribed in myoblasts, and forced expression of one of them (MyoD1) in fibroblasts
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Failed beyond 8 cell stage, probably due to an inability to activate critical
embryonic genes and dissociation of repressive epigenetic factors
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An enucleated oocyte (DNA removed) was fused with the nucleus of a skin fibroblast
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This required treatment with defined factors
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But they had very small ICMs therefore did not support ESC derivation, hence
the need for more optimisation
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These lines showed expression of pluripotency markers, sox2, Oct4 and Nanog
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Both in cell culture could form embryoid
bodies, and when injected into SCID mice, formed tumours containing cell types from all
three germ layers!
Can form human nuclear transfer ESCs from patient specific somatic cells
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H3 K9me3, 5mc
H3 K4me3, K9ac
Trying to iPSCs from differentiated cells (no need for oocyte) via viral vector transfection of
SC-associated genes
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by removing
one factor from transfection each time found which were important
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Very inefficient in human cells… only 0
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1% success rate
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Viral vectors
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Use plasmids
as non- insertional avoiding insertional mutagenesis… not as effective though
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(need improvement of this transgene-free approach)
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For cell therapy can take these cells and
repair the disease causing mutation by gene targeting
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Differentiate into patient specific
replacement of damaged cells and transplant in healthy ones
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Differentiation strategies are at present deficient… it’s hard to get the correct cell type in a
pure cell culture
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Is it
possible to model this in vitro?
Jaenisch in 2007
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Ads: Readily available
Patient specific so no transplant rejection
Used to model genetic disease
Few ethical issues
Unlimited proliferative ability
Pluripotent so can form most cell types
Title: Induced Pluripotent Stem Cells
Description: Useful short questions and answers I wrote based on UCL biomedical sciences second year Induced Pluripotent Stem Cells lecture. To get a 2.1/1st you really should be able to answer all of these.
Description: Useful short questions and answers I wrote based on UCL biomedical sciences second year Induced Pluripotent Stem Cells lecture. To get a 2.1/1st you really should be able to answer all of these.