Lecture 3: iPSCs Flashcards
Describe DNA
All cells of all organisms store their hereditary info in a double-stranded DNA genetic blueprint.
Describe mRNA
Transcribed from one strand of DNA into a complementary sequence. Provides the details on how to assemble (translate into) a protein.
Protein
comprised of covalently linked to amino acids. Does much of the work of the cell-structural functions, enzymatic signal.
What is the central dogma?
DNA > RNA> Protein
What does a promoter do?
It determines what cells will make mRNA from the gene and when.
So every cell type has some promoters that are active and some that are suppressed.
Different sets of promoters are active in pluripotent stem cells than neurons etc. (not the same as the neurons).
Explain how we can link an immature neuronal promoter (Doublecortin) to control the expression of GFP
Doublecortin is only generated when neural stem cells are generated.
You can modify the Doublecortin gene to code for a different mRNA sequence (GFP) But still have that new sequence driven by the Doublecortin promoter.
___ approaches can be used to improve the generation and purification of neurons.
Molecular
What is the FACS machine?
Fluorescent activated cell sorting of reporter lines can improve purity.
This tool works by purifying different subset of cells and splits into different channels.
Use antibody to pull the cells out.
Do your neurons have the same DNA as your skin cells.
Yes, every cell in your body has the same set of DNA and genes.
But they are different because of differential gene expression.
What makes your neurons different from your skin cells?
Different sets of genes are being expressed by skin cells vs. neurons.
Terminally differentiation (won’t become another cell).
___ determine what cells will make what specific mRNAs and when
Promoters
So every cell type has some promoters that are active and some that are suppressed.
Different sets of promoters are active in pluripotent stem cells than skin cells or neurons.
- Transcription factors control genes and different ones exist for different cell types.
The ___ promoter is only turned on and only makes Fbx15 mRNA and Fbx15 protein in a _______
Fbx15
pluripotent embryonic stem cells
It is not expressed in any differentiated cells.
Describe the modifications that can be done to the Fbx15.
You can modify the Fbx15 gene to code for a different mRNA sequence. But still have that new sequence “driven” by the Fbx15 promoter
Describe the switch between the GFP and other drug resistance proteins.
Instead of putting GFP they switched in a gene that codes for a drug resistance protein (Bgeo). The Bgeo breaks down all cells except for specific ones.
When you do this what type of cells will make this Bgeo protein?
Only cells that normally express Fbx15 pluripotent stem cells.
When you switch in a gene that codes for a drug resistance protein (Bgeo) what type of cells will not die if you treat them with that drug?
Only cells that produce the Bgeo drug resistance gene driven by Fbx15 pluripotent stem cells.
Describe what happens if you use viruses to express certain genes in fibroblasts and find that it turns on the Fbx15 promoter.
If you use viruses to express certain genes in fibroblasts and find that it turns on the Fbx15 promoter, then it suggests that those genes have made the fibroblasts more like embryonic stem cells.
The Fbx15 genes are only expressed in ____ stem cells
mouse embryonic stem cells not differentiated cells like fibroblasts so they used the Fbx15 promoter. to drive the expression of antibiotic resistance gene (bgeo).
How do you get the skin cell?
Used transcription factors in embryonic stem cells and over expressed them.
Describe the generation of iPS cells from MEF cultures via ___ factors.
24 factors that are highly expressed in pluripotent stem cells but not stem cells.
Describe the induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures defined by factors.
1.You first make Bgeo to kill off other stuff
2. Apply drug
3. Kill off skin cells.
4. Only Fbx15 ones remain.
__ factor iPSCs expressed ESC associated mRNAs and can differentiate into all __ germ lineages.
24 factors
3 germ lineages
surviving cells could become any other type of cell.
They found __ factors when each was individually removed produced no colonies at day 10
10
When these 10 factors were combined they induced more colonies than the 24 combined factors.
There are __ key factors needed
4
C-Myc, Klf4, Sox2, Oct 3/4
What types of analyses helped support the narrowing of the growth factors?
Teratoma formation confirmed production of cells from all 3 germ layers.
(Inject cells into mouse with no immune system and showed all 3 germ layers.
Microarray RNA analysis showed very similar Gene expression profiles for iPSCs and ESCs
Describe the induction of pluripotent stem cells from adult human fibroblasts defined factors.
It was found that the same factors work in humans as discovered by Takahashi and Yamanaka
