Questions for Okita et al., 2007. Generation of germline-competent induced pluripotent stem cells. Nature 448:313-318.
This paper builds on the first paper to demonstrate the technique of generating iPS cells, in 2006, from the Yamanaka lab. Some of these figures are complicated—if you find you are not sure what is happening in a figure, try to describe what was being shown in each panel, and why, and then try to draw conclusions based on what is shown. In class, we can talk about the more difficult aspects, so please make a list of the things that don’t make sense to you, so we know what to focus on in class.
1. From the introduction, what are the authors doing differently in this set of experiments than in the original paper?
2. What is Nanog (you may have to look this up separately), and what previous lines of evidence suggest that it is a good marker of stem cells?
3. Using Figure 1, describe how the authors generated Nanog iPS cells. Why do they use the GFP cassette shown? Why do they demonstrate the appearance of the GFP positive cells in the germline of the mice? What are MEF cells?
4. What were the authors demonstrating in Figure 2, and why is this figure important?
5. How did they demonstrate that the cells they had obtained were indistinguishable from ES cells (Figure 3), and what is the significance of the tumors generated in panels b and c?
6. In Figure 4, Nanog iPS and Fbx15 iPS cells are compared. Why? What do they conclude?
7. What is the significance of Figure 5?
8. Describe what has been done in Figure 6, and what can be concluded from these experiments. What is a chimaera? What happens when you cross chimaeras to normal mice in terms of what you’d expect to see in the offspring? What does this have to do with the coat color of the mice?
9. Write a one-two sentence summary of the authors conclusions.