expression of a gene in unusual place, from disease or artifically induced -introduce transgene with modifier promoter, used to understand genes function
what can a microarray tell us?
-has microscopic spots of dna with specific dna sequence (probes), probe-target hybridization is detected and quantified by chemiluminescence labeled targets -determine relative abundance of nucleic acid sequences in the target , can accomplish many genetic tests at the same time -also used to genotype mutant genomes
immunoprecipitation, look at interaction between proteins and DNA, see if specific proteins are associated with specific genomic regions (transcription factors on promoters etc), also to find specific location in genome with histone modifications -lyse cell, bind to specific dna region, immunopreciptation, protein digestion, dna purification, sent to pcr, sequencing microarray etc
what question is the boyer paper asking?
How does Oct4 regulate the pluripotency of human ES cells at the cell and molecular level?`
how did the authors of Boyer paper answer their question or prove/disprove their hypothesis?
ChIP of OCT4 followed by hybridization of genomic DNA fragments associated with OCT4 on promoter microarrays (ChIp chip) to identify promoters occupied by OCT4 and SOX2 and NANOG
why are the authors looking at Oct4, Sox2 and Nanog?
-the three co-occupy similar regions of many promoters including mRNA and miRNA target genes (*) -activate genes involved in pluipotency including chromatin regulatroy factors -repress genes invoolved in differentation of 3 germ layers -the three are master regulators of human ES cell pluipotency
what does figure 2 show?
353 protein coding genes are simultaneously bound by Oct4, sox2 and nanog, -they bind in close proximity
describe the model that boyer paper shows
in the takahashi paper why do the authors show the HDF cells in figure 2?
as a control, a and b: show that cell marker genes are not normally expressed on HDF c: show increase in expression of retroviral transgenes in hdf 6 days after transduction d:show relative global gene expression (shows expression levels of nanog, oct3/4 and sox2--expressed in iPS, ES (1:1) not in HDF)
what is the take home message of figure 2?
human iPS cells are similar to ES cells in cell marker genes and global gene expression
describe figure 5
data demonstrated that iPS cells could differentiate into three germ layers in vitro.
what might figure 7 tell us about the use of these cells in humans at this time?
teratomas are human tumors so adding these cells to patients may induce tumor formation and iPOS cells are similar but not identical to ES cells, still need human ES cell work
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