Dry Lab 1

Question 1

Why did the researchers conduct their study?

Answer 1

• 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

Question 2

Why is the research important?

Answer 2

• 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.

Question 3

What is the assay they used?

Answer 3

• 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.

Question 4

How did they chose their factors?

Answer 4

• 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• Withdrawn individual factors to determine 10 as critical.
• Withdrawn individual factors to determine 4 as critical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

Question 5

What characteristics of ES cells did the iPS cells express?

Answer 5

• morphologically similar: round, large nucleoli, scant cytoplasm
• proliferation rates
• expressed ES cell markers

Question 6

What four factors were found to be the most critical?

Answer 6

• Oct3/4
• Sox2
• c-Myc
• Klf4

Question 7

What is one control they used?

Answer 7

• 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.