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hPGK1        eGFP-Neo26 8 3   PolyA        Ins        PolyA        mCherry                   Jay Gantz, Laflamme Lab Meetin...
What I told you on March 2nd...EF1a-CRE lenti plasmid is done, need to make            virus, titer and test CK7-CRE seeme...
Let’s start with the cloning7      4               5+6                       8 3              2        1                  ...
Let’s start with the cloning7      4               5+6                       8 3              2        1                  ...
Let’s start with the cloning7      4               5+6                       8 3              2        1                  ...
Let’s start with the cloning7      4               5+6                       8 3              2        1                  ...
Solution:7      4             5+6                       8 3              2        1                                       ...
Solution: A Patch!7      4             5+6                       8 3              2        1                              ...
Solution: A Patch!7      4             5+6                  2        1                    2A      CK7Ins                  ...
Solution: A Patch!7      4             5+6                                        2        1                              ...
Solution: A Patch!7      4             5+6                                        2        1                              ...
Meanwhile... tested to look for GFP7      4               5+6                       8 3              2        1           ...
Meanwhile... tested to look for GFP7      4               5+6                       8 3              2        1           ...
Meanwhile... tested to look for GFP7      4               5+6                       8 3              2        1           ...
Why no green?7      4             5+6                       8 3              2        1                                   ...
Why no green?7      4               5+6                       8 3              2        1                                 ...
Why no green?7      4               5+6                       8 3              2        1                                 ...
eGFP-Neo Advertised as a fusion protein.Looking closer at the sequence:  This is not a fusion protein.     Called OligoEng...
Solution:            8
Solution: eGFP-Zeo!             PLoS One. 2009; 4(8): e6529.A Versatile Viral System for Expression andDepletion of Protei...
Final construct should look like:                    2A      CK7Ins                                            eGFP-Zeo   ...
Final construct should look like:                                       PolyA                                             ...
CK7-CRE IHC     10
Last time proposed some terrible ideas:       Dish 1                        Dish 2Mouse anti-tdTomato             Mouse an...
But I showed you that we had good red/green after fixation                           12
First Pass: Endogenous tdTomato   Endogenous eGFP     Mouse anti-cTnTanti-Mouse IgG Alexa Blue Biotin-Mouse anti-CRE   Str...
14
Second Pass:Endogenous tdTomato           Endogenous tdTomato  Endogenous eGFP               Endogenous eGFP   Mouse anti-...
16
17
Next IHC approach:Redo with negative CRE controlsLet red/green go longer (14 days)               18
EF1a-CRE LentiTrial to see if it works: throw it onto mTmG undiffs   Positive control of CK7-CRE (has some leak)          ...
/01(2132$45367$             $%."             $%+"             $%"             $%*"!"#$%&()$                         !"#"$%...
Source of EF1a-CRE problem?A: dirty DNA leading to empty or non-functional viruses             B: Bad sequence or backbone...
March 2011Questions?
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2011 4.14

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2011 4.14

  1. 1. hPGK1 eGFP-Neo26 8 3 PolyA Ins PolyA mCherry Jay Gantz, Laflamme Lab Meeting April 14, 2011
  2. 2. What I told you on March 2nd...EF1a-CRE lenti plasmid is done, need to make virus, titer and test CK7-CRE seemed to work but IHC needed Cloning: problems with restriction sites (dammethylation), several lucky breaks, one last step 2
  3. 3. Let’s start with the cloning7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Thought there was one last step: 2nd insulator 3
  4. 4. Let’s start with the cloning7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Thought there was one last step: 2nd insulator However: cut site between pAs doesn’t cut. Uh oh. 3
  5. 5. Let’s start with the cloning7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Thought there was one last step: 2nd insulator However: cut site between pAs doesn’t cut. Uh oh. No other sites. No obvious options. 3
  6. 6. Let’s start with the cloning7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Thought there was one last step: 2nd insulator However: cut site between pAs doesn’t cut. Uh oh. No other sites. No obvious options. Tried sequencing between PolyAs: nada 3
  7. 7. Solution:7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry 4
  8. 8. Solution: A Patch!7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry 4
  9. 9. Solution: A Patch!7 4 5+6 2 1 2A CK7Ins eGFP-Neo hPGK PuroR mCherry 4
  10. 10. Solution: A Patch!7 4 5+6 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Patch Patch advantages: •Perfect sequence (we ordered the oligo) •Engineered multiple new and unique restriction sites around all components 4
  11. 11. Solution: A Patch!7 4 5+6 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Patch Patch advantages: •Perfect sequence (we ordered the oligo) •Engineered multiple new and unique restriction sites around all componentsPatch disadvantage: Time (ordered mid-March, any day now...) 4
  12. 12. Meanwhile... tested to look for GFP7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Idea: check if right half is functional in HEKs 5
  13. 13. Meanwhile... tested to look for GFP7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Idea: check if right half is functional in HEKs Transfected (along with a + control) 5
  14. 14. Meanwhile... tested to look for GFP7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Idea: check if right half is functional in HEKs Transfected (along with a + control) No green. 5
  15. 15. Why no green?7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Reason 1: low quality DNA prep (P/C made a huge difference for Nathan) 6
  16. 16. Why no green?7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Reason 1: low quality DNA prep (P/C made a huge difference for Nathan) Reason 2: mutation? but we have ok sequence 6
  17. 17. Why no green?7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Reason 1: low quality DNA prep (P/C made a huge difference for Nathan) Reason 2: mutation? but we have ok sequence Reason 3: look into the construct further... 6
  18. 18. eGFP-Neo Advertised as a fusion protein.Looking closer at the sequence: This is not a fusion protein. Called OligoEngine... 7
  19. 19. Solution: 8
  20. 20. Solution: eGFP-Zeo! PLoS One. 2009; 4(8): e6529.A Versatile Viral System for Expression andDepletion of Proteins in Mammalian Cells Campeau et al. A true fusion protein! 8
  21. 21. Final construct should look like: 2A CK7Ins eGFP-Zeo hPGK PuroR mCherry 9
  22. 22. Final construct should look like: PolyA PolyA 2A CK7Ins eGFP-Zeo hPGK mCherry Ins PuroR Patch 9
  23. 23. CK7-CRE IHC 10
  24. 24. Last time proposed some terrible ideas: Dish 1 Dish 2Mouse anti-tdTomato Mouse anti-CRE ~$140 ~$250anti-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-cTnTanti-Chicken IgY YFP anti-Chicken IgY YFPHoechst Counterstain Hoechst Counterstain 11
  25. 25. But I showed you that we had good red/green after fixation 12
  26. 26. First Pass: Endogenous tdTomato Endogenous eGFP Mouse anti-cTnTanti-Mouse IgG Alexa Blue Biotin-Mouse anti-CRE Strep 647 Far Red 13
  27. 27. 14
  28. 28. Second Pass:Endogenous tdTomato Endogenous tdTomato Endogenous eGFP Endogenous eGFP Mouse anti-cTnT Mouse anti-CREanti-Mouse IgG Far Red anti-Mouse IgG Far Red Dapi Dapi 15
  29. 29. 16
  30. 30. 17
  31. 31. Next IHC approach:Redo with negative CRE controlsLet red/green go longer (14 days) 18
  32. 32. EF1a-CRE LentiTrial to see if it works: throw it onto mTmG undiffs Positive control of CK7-CRE (has some leak) No Green! 19
  33. 33. /01(2132$45367$ $%." $%+" $%" $%*"!"#$%&()$ !"#"$%$$&(")"$%$*+*" $%2" ,-"#"$%.../0" $%/" $%&" $%1" $%0" $" $" 2$" 0$$" 02$" 1$$" 12$" *+%$(,-).$ 20
  34. 34. Source of EF1a-CRE problem?A: dirty DNA leading to empty or non-functional viruses B: Bad sequence or backbone I did a P/C got clean DNA and submitted for full sequencing 21
  35. 35. March 2011Questions?

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