5. Alternatively, induce with 0.1 mM IPTG overnight at 16
C.
6. In this step, it is not necessary to lyse the pellet thoroughly
because the sonication procedure in the next step will disrupt it
completely.
7. The sonication will produce the heat leading the degradation of
proteins, therefore, this procedure should be done with cooling by ice.
8. Alternatively, the resin can be transferred to a disposable plastic
column and then washed and eluted on the column via gravityflow procedures.
9. Total time of dialysis should be between 6 and 8 h because long
hours of it often cause a precipitation of proteins.
10. The backbone vector of mouse MafA-11R plasmid is pGEX6p-1. It contains the recognition sequence for PreScission
protease between the GST domain and the sequence encoding
MafA-11R.
11. Overnight incubation can be performed if desired.
12. Gelatin solution should be aspirated before plating the feeder
cells.
13. Treatment with mitomycin C should be performed at a concentration of 10 μg/mL for 2–3 h at 37
C in a CO 2 incubator.
Alternatively, those cells are commercially available.
14. We have tested the mouse ESC lines E14tg2a (provided by the
RIKEN BRC) [16], Sk7 (carrying a Pdx1-promoter-driven
GFP reporter transgene and provided by Dr. Shoen Kume)
[17, 18] and ING112 (carrying a Ins1-promoter-driven GFP
reporter transgene and provided by Dr. Shoen Kume)
[19, 20].
15. Overconfluent culturing should be avoided because it leads to
differentiation of the ESCs.
16. The coating of the synthetic fiber plate with PLL increase the
amount of absorbed laminin to the plate [7].
17. The coating of the synthetic fiber plate with laminin facilitates
the differentiation of mouse ESCs to pancreatic lineages [6, 7].
18. The total number of cells would be 5–10 Â 10
6 from one
60 mm dish.
19. The cells should be resuspended in 1 mL of plating medium
with a P1000 micropipette and then resuspended in a defined
volume of the medium with a 10 mL pipette.
20. Multi-channel micropipette is convenient for plating the cells
and changing the media.
21. The differentiation medium should be warmed at 37
C in a
water bath before use.
94
Taku Kaitsuka and Kazuhito Tomizawa
C.
6. In this step, it is not necessary to lyse the pellet thoroughly
because the sonication procedure in the next step will disrupt it
completely.
7. The sonication will produce the heat leading the degradation of
proteins, therefore, this procedure should be done with cooling by ice.
8. Alternatively, the resin can be transferred to a disposable plastic
column and then washed and eluted on the column via gravityflow procedures.
9. Total time of dialysis should be between 6 and 8 h because long
hours of it often cause a precipitation of proteins.
10. The backbone vector of mouse MafA-11R plasmid is pGEX6p-1. It contains the recognition sequence for PreScission
protease between the GST domain and the sequence encoding
MafA-11R.
11. Overnight incubation can be performed if desired.
12. Gelatin solution should be aspirated before plating the feeder
cells.
13. Treatment with mitomycin C should be performed at a concentration of 10 μg/mL for 2–3 h at 37
C in a CO 2 incubator.
Alternatively, those cells are commercially available.
14. We have tested the mouse ESC lines E14tg2a (provided by the
RIKEN BRC) [16], Sk7 (carrying a Pdx1-promoter-driven
GFP reporter transgene and provided by Dr. Shoen Kume)
[17, 18] and ING112 (carrying a Ins1-promoter-driven GFP
reporter transgene and provided by Dr. Shoen Kume)
[19, 20].
15. Overconfluent culturing should be avoided because it leads to
differentiation of the ESCs.
16. The coating of the synthetic fiber plate with PLL increase the
amount of absorbed laminin to the plate [7].
17. The coating of the synthetic fiber plate with laminin facilitates
the differentiation of mouse ESCs to pancreatic lineages [6, 7].
18. The total number of cells would be 5–10 Â 10
6 from one
60 mm dish.
19. The cells should be resuspended in 1 mL of plating medium
with a P1000 micropipette and then resuspended in a defined
volume of the medium with a 10 mL pipette.
20. Multi-channel micropipette is convenient for plating the cells
and changing the media.
21. The differentiation medium should be warmed at 37
C in a
water bath before use.
94
Taku Kaitsuka and Kazuhito Tomizawa