Dry Lab 1 Flashcards
1
Q
Why did the researchers conduct their study?
A
- It is known that cells can enter an embryonic-like state from their contents being moved into a oocyte or from being fused to an embryonic stem cell.
- It is not known what factors cause this change
2
Q
Why is the research important?
A
- ES cells can be used to treat diseases however it is unethical to use human embryos.
- If a way to produce embryonic-like stem cells is found, these treatments can still be developed.
3
Q
What is the assay they used?
A
- Developed an assay system in which a pluripotent state can be detected by resistance to G418.
- Inserted a Bgeo cassette
- ES cells activate the FBx15 locus.
- ES cells are resistant to high concentrations of G418.
- Somatic cells are sensitive to normal concentrations of G418.
4
Q
How did they chose their factors?
A
- Started with 24 factors.
- Introduced to mouse embryonic fibroblasts by retroviral transduction.
- No single factor produced G418 resistance, however all 24 together did.
- Withdrawn individual factors to determine 10 as critical.
- Withdrawn individual factors to determine 4 as critical
- Loss of function experiment
5
Q
What characteristics of ES cells did the iPS cells express?
A
- morphologically similar: round, large nucleoli, scant cytoplasm
- proliferation rates
- expressed ES cell markers
6
Q
What four factors were found to be the most critical?
A
- Oct3/4
- Sox2
- c-Myc
- Klf4
7
Q
What is one control they used?
A
- They compared colony numbers between different types of colonies based on the factors added and had a mock colony which had no factors added. This is a negative control since it should not produce a colony.
