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hPGK
1
        eGFP-Neo
2
6 8 3   PolyA
        Ins
        PolyA
        mCherry



                   Jay Gantz, Laflamme Lab Meeting
                            April 14, 2011
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 (dam
methylation), several lucky breaks, one last step




                        2
Let’s start with the cloning

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               eGFP-Neo   hPGK




                                                 Ins
              PuroR        mCherry



           Thought there was one last step: 2nd insulator




                                     3
Let’s start with the cloning

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               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
Let’s start with the cloning

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               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
Let’s start with the cloning

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               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
Solution:
7      4             5+6                       8 3              2        1




                                       PolyA



                                                     PolyA
                    2A
      CK7
Ins



                                                             eGFP-Neo   hPGK




                                               Ins
            PuroR        mCherry




                                   4
Solution: A Patch!
7      4             5+6                       8 3              2        1




                                       PolyA



                                                     PolyA
                    2A
      CK7
Ins



                                                             eGFP-Neo   hPGK




                                               Ins
            PuroR        mCherry




                                   4
Solution: A Patch!
7      4             5+6                  2        1


                    2A
      CK7
Ins



                                       eGFP-Neo   hPGK
            PuroR        mCherry




                                   4
Solution: A Patch!
7      4             5+6                                        2        1




                                       PolyA



                                                     PolyA
                    2A
      CK7
Ins



                                                             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
Solution: A Patch!
7      4             5+6                                        2        1




                                       PolyA



                                                     PolyA
                    2A
      CK7
Ins



                                                             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
Patch disadvantage: Time (ordered mid-March, any
                    day now...)
                                   4
Meanwhile... tested to look for GFP

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               eGFP-Neo   hPGK




                                                 Ins
              PuroR        mCherry



            Idea: check if right half is functional in HEKs




                                     5
Meanwhile... tested to look for GFP

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               eGFP-Neo   hPGK




                                                 Ins
              PuroR        mCherry



            Idea: check if right half is functional in HEKs
                Transfected (along with a + control)




                                     5
Meanwhile... tested to look for GFP

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               eGFP-Neo   hPGK




                                                 Ins
              PuroR        mCherry



            Idea: check if right half is functional in HEKs
                Transfected (along with a + control)
                              No green.



                                     5
Why no green?

7      4             5+6                       8 3              2        1




                                       PolyA



                                                     PolyA
                    2A
      CK7
Ins




                                                             eGFP-Neo   hPGK




                                               Ins
            PuroR        mCherry


      Reason 1: low quality DNA prep (P/C made a huge
                    difference for Nathan)




                                   6
Why no green?

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               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
Why no green?

7      4               5+6                       8 3              2        1




                                         PolyA



                                                       PolyA
                      2A
      CK7
Ins




                                                               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
eGFP-Neo

 Advertised as a fusion protein.
Looking closer at the sequence:




  This is not a fusion protein.
     Called OligoEngine...



                7
Solution:




            8
Solution: eGFP-Zeo!




             PLoS One. 2009; 4(8): e6529.



A Versatile Viral System for Expression and
Depletion of Proteins in Mammalian Cells
               Campeau et al.

 A true fusion protein!

                          8
Final construct should look like:

                    2A
      CK7
Ins




                                            eGFP-Zeo   hPGK
            PuroR        mCherry




                                   9
Final construct should look like:




                                       PolyA



                                                     PolyA
                    2A
      CK7
Ins




                                                             eGFP-Zeo   hPGK
                         mCherry




                                               Ins
            PuroR

                                           Patch




                                   9
CK7-CRE IHC




     10
Last time proposed some terrible ideas:

       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

                        11
But I showed you that we had good red/green after fixation




                           12
First Pass:

 Endogenous tdTomato
   Endogenous eGFP
     Mouse anti-cTnT
anti-Mouse IgG Alexa Blue
 Biotin-Mouse anti-CRE
   Strep 647 Far Red



           13
14
Second Pass:


Endogenous tdTomato           Endogenous tdTomato
  Endogenous eGFP               Endogenous eGFP
   Mouse anti-cTnT                Mouse anti-CRE
anti-Mouse IgG Far Red        anti-Mouse IgG Far Red
        Dapi                          Dapi


                         15
16
17
Next IHC approach:
Redo with negative CRE controls
Let red/green go longer (14 days)




               18
EF1a-CRE Lenti


Trial to see if it works: throw it onto mTmG undiffs


   Positive control of CK7-CRE (has some leak)


                    No Green!




                         19
/01(2132$45367$
             $%."

             $%+"

             $%'"

             $%*"
!"#$%&'()$




                         !"#"$%$$&'(")"$%$*+*"
             $%2"
                             ,-"#"$%.../0"
             $%/"

             $%&"

             $%1"

             $%0"

               $"
                    $"             2$"           0$$"                02$"   1$$"   12$"
                                                        *+%$(,-).$




                                                         20
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
March 2011



Questions?

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2011 4.14

  • 1. hPGK 1 eGFP-Neo 2 6 8 3 PolyA Ins PolyA mCherry Jay Gantz, Laflamme Lab Meeting April 14, 2011
  • 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 (dam methylation), several lucky breaks, one last step 2
  • 3. Let’s start with the cloning 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry Thought there was one last step: 2nd insulator 3
  • 4. Let’s start with the cloning 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins 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. Let’s start with the cloning 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins 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. Let’s start with the cloning 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins 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. Solution: 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry 4
  • 8. Solution: A Patch! 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry 4
  • 9. Solution: A Patch! 7 4 5+6 2 1 2A CK7 Ins eGFP-Neo hPGK PuroR mCherry 4
  • 10. Solution: A Patch! 7 4 5+6 2 1 PolyA PolyA 2A CK7 Ins 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. Solution: A Patch! 7 4 5+6 2 1 PolyA PolyA 2A CK7 Ins 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 Patch disadvantage: Time (ordered mid-March, any day now...) 4
  • 12. Meanwhile... tested to look for GFP 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry Idea: check if right half is functional in HEKs 5
  • 13. Meanwhile... tested to look for GFP 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry Idea: check if right half is functional in HEKs Transfected (along with a + control) 5
  • 14. Meanwhile... tested to look for GFP 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry Idea: check if right half is functional in HEKs Transfected (along with a + control) No green. 5
  • 15. Why no green? 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins eGFP-Neo hPGK Ins PuroR mCherry Reason 1: low quality DNA prep (P/C made a huge difference for Nathan) 6
  • 16. Why no green? 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins 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. Why no green? 7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7 Ins 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. eGFP-Neo Advertised as a fusion protein. Looking closer at the sequence: This is not a fusion protein. Called OligoEngine... 7
  • 20. Solution: eGFP-Zeo! PLoS One. 2009; 4(8): e6529. A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells Campeau et al. A true fusion protein! 8
  • 21. Final construct should look like: 2A CK7 Ins eGFP-Zeo hPGK PuroR mCherry 9
  • 22. Final construct should look like: PolyA PolyA 2A CK7 Ins eGFP-Zeo hPGK mCherry Ins PuroR Patch 9
  • 24. Last time proposed some terrible ideas: 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 11
  • 25. But I showed you that we had good red/green after fixation 12
  • 26. First Pass: Endogenous tdTomato Endogenous eGFP Mouse anti-cTnT anti-Mouse IgG Alexa Blue Biotin-Mouse anti-CRE Strep 647 Far Red 13
  • 27. 14
  • 28. Second Pass: Endogenous tdTomato Endogenous tdTomato Endogenous eGFP Endogenous eGFP Mouse anti-cTnT Mouse anti-CRE anti-Mouse IgG Far Red anti-Mouse IgG Far Red Dapi Dapi 15
  • 29. 16
  • 30. 17
  • 31. Next IHC approach: Redo with negative CRE controls Let red/green go longer (14 days) 18
  • 32. EF1a-CRE Lenti Trial to see if it works: throw it onto mTmG undiffs Positive control of CK7-CRE (has some leak) No Green! 19
  • 33. /01(2132$45367$ $%." $%+" $%'" $%*" !"#$%&'()$ !"#"$%$$&'(")"$%$*+*" $%2" ,-"#"$%.../0" $%/" $%&" $%1" $%0" $" $" 2$" 0$$" 02$" 1$$" 12$" *+%$(,-).$ 20
  • 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

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