2011 4.14
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2011 4.14

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2011 4.14 2011 4.14 Presentation Transcript

  • hPGK1 eGFP-Neo26 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 (dammethylation), several lucky breaks, one last step 2
  • 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
  • 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
  • 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
  • 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
  • Solution:7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry 4
  • Solution: A Patch!7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry 4
  • Solution: A Patch!7 4 5+6 2 1 2A CK7Ins eGFP-Neo hPGK PuroR mCherry 4
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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 andDepletion of Proteins in Mammalian Cells Campeau et al. A true fusion protein! 8
  • Final construct should look like: 2A CK7Ins eGFP-Zeo hPGK PuroR mCherry 9
  • Final construct should look like: PolyA PolyA 2A CK7Ins eGFP-Zeo hPGK mCherry Ins PuroR Patch 9
  • CK7-CRE IHC 10
  • 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
  • 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 Strep 647 Far Red 13
  • 14
  • 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
  • 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) 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 2011Questions?