1. Jay Gantz, Laflamme Lab Meeting
January 13, 2011

2. What I told you on January 13th...
7 4 5 6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Shouldn’t be long now... 3 more easy steps
Exposing a beating mTmG syncytium to CK7-CRE lenti is
problematic from a visualization standpoint: replate and
transduce single cells
Will make an EF1a-CRE lenti as a control for mTmG
2

3. What I told you on January 13th...
7 4 5 6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Shouldn’t be long now... 3 more easy steps
Exposing a beating mTmG syncytium to CK7-CRE lenti is
problematic from a visualization standpoint: replate and
transduce single cells
Will make an EF1a-CRE lenti as a control for mTmG
2

4. EF1a-CRE lenti plasmid is done.
Virus production is in process with Kara’s help.
3

5. Beating mTmG cells were successfully replated.
CK7-CRE lenti exposure worked as expected.
Cells made full red to green transition in ~10-14 days.
Need to order antibodies for staining.
4

6. Staining Plan for mTmG Cells... help?
Dish 1 Dish 2
Mouse anti-tdTomato Mouse anti-CRE
~$140 ~$250
anti-Mouse IgG RFP anti-Mouse IgG RFP
Rabbit anti-GFP Rabbit anti-GFP
anti-Rabbit IgG GFP anti-Rabbit IgG GFP
Chicken anti-cTnT ~$325 Chicken anti-cTnT
anti-Chicken IgY YFP anti-Chicken IgY YFP
Hoechst Counterstain Hoechst Counterstain
5

7. But do I actually need to stain for tdTomato and eGFP?
eGFP
Alive After 5’ Triton + 5’ Hoescht
After 5’ 4% PF
raw photos 2s exposure
6

8. But do I actually need to stain for tdTomato and eGFP?
tdTomato
Alive After 5’ Triton + 5’ Hoescht
After 5’ 4% PF
7

9. Merge + Levels adjustment
Alive
After 5’ Triton + 5’ Hoescht
After 5’ 4% PF
8

10. So.... cloning.
7 4 5 6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
9

11. So.... cloning.
7 4 5 6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Step 6.... just.... didn’t.... work.
9

12. So.... cloning.
7 4 5 6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Step 6.... just.... didn’t.... work.
9

13. So.... cloning.
7 4 5 6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Step 6.... just.... didn’t.... work.
New Plan: cut out step 5
replace with new 5+6 PCR from John Mignone
9

14. So.... cloning.
7 4 8 3 2 1
PolyA
CK7
Ins
eGFP-Neo hPGK
Ins
Step 6.... just.... didn’t.... work.
New Plan: cut out step 5
replace with new 5+6 PCR from John Mignone
9

15. So.... cloning.
7 4 5+6 8 3 2 1
PolyA
PolyA
2A
CK7
Ins
eGFP-Neo hPGK
Ins
PuroR mCherry
Step 6.... just.... didn’t.... work.
New Plan: cut out step 5
replace with new 5+6 PCR from John Mignone
9

16. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
10

17. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
Homology Arm Cut 1 Homology Arm
10

18. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
Homology Arm Cut 1 Homology Arm
Homology Arm Insert 1 Homology Arm
10

19. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
Homology Arm Cut 1 Homology Arm
Homology Arm Insert 1 Homology Arm
Cut 2
Homology Arm Insert 1 Homology Arm
10

20. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
Homology Arm Cut 1 Homology Arm
Homology Arm Insert 1 Homology Arm
Cut 2
Homology Arm Insert 1 Homology Arm
Homology Arm Insert 2 Insert 1 Homology Arm
etc...
10

21. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
10

22. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
4 5 6
EcoRI
PolyA
2A
Homology Arm CK7 PuroR mCherry
7 4 5 6
PolyA
2A
CK7
Ins
Homology Arm PuroR mCherry
10

23. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
Solution 4 5 6
EcoRI
PolyA
2A
Homology Arm CK7 PuroR mCherry
7 4 5 6
PolyA
2A
CK7
Ins
Homology Arm PuroR mCherry
10

24. It worked... BUT
Problem 1
5+6
EcoRI
PolyA
2A
PuroR mCherry
Solution 4 5 6
HindIII
EcoRI
PolyA
2A
Homology Arm CK7 PuroR mCherry
7 4 5 6
PolyA
2A
CK7
Ins
Homology Arm PuroR mCherry
10

25. Problem 2
5+6
PolyA
XbaI
2A
PuroR mCherry
11

26. Problem 2
5+6
PolyA
XbaI
2A
PuroR mCherry
7 4 5 6
PolyA
XbaI
XbaI
2A
CK7
Ins
Homology Arm PuroR mCherry
PolyA
XbaI
XbaI
2A
cGata6
Ins
Homology Arm PuroR mCherry
11

27. Dam methylation
Dam methylase recognizes:
5’-GATC-3’ 5’-GAmTC-3’
12

28. Dam methylation
Dam methylase recognizes:
5’-GATC-3’ 5’-GAmTC-3’
Xba1:
5’...TCTAGA...3’
3’...AGATCT...5’
12

29. Dam methylation
Dam methylase recognizes:
5’-GATC-3’ 5’-GAmTC-3’
Xba1:
5’...TCTAGA...3’
3’...AGATCT...5’
5’...GATCTAGA...3’
3’...CTAGATCT...5’
12

30. Dam methylation
Dam methylase recognizes:
5’-GATC-3’ 5’-GAmTC-3’
Xba1:
5’...TCTAGA...3’
3’...AGATCT...5’
5’...GATCTAGA...3’ 5+6
PolyA
XbaI
2A
3’...CTAGATCT...5’ PuroR mCherry
12

31. Solution:
Grow plasmid in dam+ bugs
Have verified that I only get the CK7 cut out
13

32. February 2011
Questions?