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tophat and pptx questions
major hypothesis of paper 2
the same four yamanaka factors that can induce a pluripotent state in adult mouse cells will induce a pluripotent state in adult human cells
what is transdifferentiation?
cells regress to a point (pluripotent) when they can switch lineages
what is reprogramming?
aims to induce differentiated cells into reverting to pluripotency, from which they can differentiate into almost any cell type (i.e. transdifferentiation)
what is transduction?
process of stably introducing genetic material into cells (genes in the genome)
what type of animal did takahashi and yamanaka generate?
a transgenic mouse that had a transgene designed to select for ES-like cells
FBx15 promoter
only active in early embryo and ES cells
beta-galactosidase
generates blue color with X-gal substratene
neomycin resistant protein (geo)
renders cells resistant to neomycin
beta-geo
encodes a beta-galactosidase-neomycin resistant fusion protein
what are the four genes found to induce pluripotency?
oct 3/4, sox2, c-myc, and Klf4
what type of cell are the researchers starting with?
human dermal fibroblasts (HDF) - skin cells
how were the pluripotent genes put into HDF cells?
via 4 different retroviruses
why were the transformed HDF cells plated on mitomycin C-treated feeder layers of cells?
mitomycin C damages DNA and prevents DNA replication in the feeder cells
what is the purpose of feeder cells?
to provide growth factors
what are retroviruses?
organisms that use RNA as its genetic material, converts retroviral RNA into DNA
retroviruses have the ability to do what with their ssRNA genome
transform it into dsDNA
what makes retroviruses a powerful gene delivery tool?
genetic stability, high expression, and flexible genome
what special protein do retroviruses contain to achieve their goal?
reverse transcriptase
what do the long terminal repeats (LTR) in the genome provide?
promoter for transcription, sequences for recombination into the genome, and sequences for packaging RNA into a virus particle
Gag
poly protein, gag = group antigens gene; capsid protein
Pol
reverse transcriptase gene
env
envelope protein gene
how is the retrovirus engineered?
by substituting gag, pol, and env w the sequence of interest, and moving the three genes somewhere else in the genome! thus the LTR will be packaged into the virus particle, but not the genes for gag, pol, and env.
how is the mRNA processed?
polyadenylated and processed using 3’ LTR signals
what do virus packaging cell lines do for the retrovirus
provide the packaging proteins gag-pol-envw
what does the transfer plasmid do?
provides the RNA encoding the gene of interest!
what gives the retrovirus its’ specificity?
the env protein produced by env gene; each env protein is specific to the host cell type
ecotrophic particles
can only infect mouse or rat cells
amphotropic particles
can infect more diverse mammals, including humans
how did the researchers optimize efficiency of retroviral transduction into adult human dermal fibroblasts (HDF)?
mouse receptor Slc7a1 was introduced into the HDF cells; provides specificity to the env protein (results in high transduction efficiency)
LTR
promoter for transcription of retroviral genes
psi
packaging signal
FBS (fetal bovine serum)
provides growth factors that nurture the adult dermal cellsf
feeder cells
layer of cells unable to divide (due to mitomycin damaging their DNA), provides extracellular secretions to help another cell proliferate while keeping them undifferentiated
ES
embryonic cell culture medium
bFGF
multifunctional cell growth factor that stimulates the growth of cells derived from mesodermal and neuroectodermal cells
what characterizes the iPS cells? (physically)
large nuclei and scant cytoplasm
what characterizes ES-like cells? (physically)
flat colonies, with differentiated cells in the middle
what are ES cell specific markers?
SSEA-3 and SSEA-4
what are tumor related antigens?
TRA-1-60, TRA-1-81 and TRA-2-49/6E
what is NANOG?
DNA binding homeobox transcription factor involved in ES cell proliferation, renewal, and pluripotency
which of these pictures shows ES cell surface markers?
I, J, K, L, M, and N (NOT H)
what techniques were used to confirm that human iPS express ES genes?
RT-PCR and Western Blot
Which TFs are specific to iPS cells?
Oct 3/4, Sox2, hTERT, UTF1, KLF4, and c-MYC
why is it so important that the hTERT transcription factor be expressed highly in iPS cells?
because its related to the telomerase gene! stem cells need to maintain their telomerase length such they can differentiate and survive
do adult cells that replicate, but are not stem cells, have high or low telomerase activity?
low telomerase activity, contributes to the mortality of the cells and limits their replicative potentialwh
what specific cells often exhibit high telomerase activity?
Cancer cells
how did the authors demonstrate that the iPS cells of HDF origin resemble ES cells?
A. immunohistory using ES markers
B. miRNA profiling
C. chromatin-immunoprecipitation
D. detected transcripts of ES cell-marker genes
A. and D.
what did quantitative PCR indicate for the retroviral genes?
that they were silenced!
what technique was used to measure the similarity of iPS cells to hES cells?
DNA microarrays - outdated process (use RNA-seq instead)
microarrays provide one way to assess relative levels of expression of all RNAs in cells. Which of the following is a way to alternatively acquire this info?
A. qPCR
B. northern blot
C. ChiP-seq
D. southern blot
E. RNA-seq
E. RNA-seq
how does microarray hybridization work?
mRNA is extracted, the cDNA is prepped by reverse transcription, which is used to produce cRNA via in vitro transcription during which cRNA is labeled with fluorescence
what unique characteristic do promoters of ES cell-specific genes have?
presence or absence of DNA methylation, as well as patterns of histone post-translational modifications
how was methylation detected in the iPS cells?
via bisulfite sequencing; accurate to the single base pair resolution
which types of post-translational modifications or patterns of DNA methylation activate or inactivate expression
acetylation occurs at lysine residues and activates genes
methylation can activate or inactivate: K4 activates, K27 inactivates
what occurs to the DNA after treatment with sodium bisulfite?
conversion of unmethylated cytosine into uracil via deamination
what are the steps of bisulfite sequencing?
DNA is denatured
treatment with sodium bisulfite to convert unmethylated C to U
PCR occurs with primers specific to one of two modified chains
resulting ampificate is sequenced immediately or after preliminary cloning
select all that are correct about bisulfite sequencing
A. process to detect all histone markers
B. process that can be used to detect and analyze methylation markers at a single bp resolution
C. denatured DNA is treated with sodium bisulfite
D. unmethylated C is converted to U
E. unmethylated C is inverted to T
B. C. and D.
what happens to oct 3/4 and nanog promoters in iPS cells?
they remain unmethylated (aka active!!)
what happens to oct3/4 and nanog promoters in parental HDF cells?
they’re methylated (aka inactive)
how do genes appear on ChiP-seq that are linked to pluripotency?
active marks in iPS cells, but repressive marks in adult fibroblasts
how do the differentiated genes appear in ChiP-seq?
very low, but in a bivalent state = cells aren’t differentiated YET (but they can be!)
identify the markers for cell differentiation
A. Gata6
B. MSX2
C. Nat1
D. CDX2
A. and B.
how did the authors confirm they had created stem cells, and not kept them in iPS state?
via RT-PCR; showed expression of the 3 germ cell lineages and no expression in undifferentiated state (i.e. iPS cells)
why did the authors include the no RT control lane in the RT-PCR?
to confirm the template is cDNA and not genomic DNA
what was a test the researchers did with mice to prove the iPS cells could differentiate?
injected human iPS cells into SCID mice - 9 wks later, tumors formed!
what is the long term fate of the retroviral constructs in the cells?
A. expressed continuously
B. silenced in the long term
Silenced in the long term
Note
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