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Transfection of alternative cell lines

Transfection of alternative cell lines. RAW 264.7 3T3-L1 J774 AtT-20. Expression of YFP in Electroporated WEHI 231 cells. Phase. CMV_YFP. 30-60% transfection efficiency. Expression of YFP in electroporated WEHI 231 and HEK293 cells. WEHI 231 293.

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Transfection of alternative cell lines

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  1. Transfection of alternative cell lines RAW 264.7 3T3-L1 J774 AtT-20

  2. Expression of YFP in Electroporated WEHI 231 cells Phase CMV_YFP 30-60% transfection efficiency

  3. Expression of YFP in electroporated WEHI 231 and HEK293 cells WEHI 231 293 YFP positive control Ctrl YFP1 YFP2 Ctrl YFP1 YFP2 M KDa 63.8 49.5 37.4 YFP 26.0 20.5 Anti-GFP (JL-8) blot Total cell lysate from ~1 mil cells per lane

  4. Expression of YFP-Akt in Electroporated WEHI 231 cells Phase EF1_YFP-Akt ~30% transfection efficiency

  5. Expression of YFP-Akt in electroporated WEHI 231 and HEK293 cells WEHI-231 293 YFP positive control Ctrl YFP-Akt Ctrl YFP-Akt KDa M 182.9 113.7 80.9 YFP-Akt 63.8 49.5 37.4 YFP 26.0 20.5 Anti-GFP (JL-8) blot Total cell lysate from ~1 mil cells per lane

  6. Expression of FLAG-Stat3 in retrovirus infected and selected WEHI 231 M TCL IP 120.0 FLAG-Stat3 83.8 65.8 Anti-Flag Blot ~200,000 cells in TCL lane ~24 mil cells in IP lane

  7. Lipofectamine2000 FuGene6 Effectene SuperFect GenePorter2 Electroporation Retrovirus infection CMV_YFP EF1_YFP Transfection Tests on Alternative Cells

  8. RAW264.7 Transfection Summary

  9. Lipofection of RAW 264.7 5 ug DNA, 10 ul of Lipofectamine2000 (6-well plate) Phase CMV_YFP Phase EF-1_YFP

  10. Lipofection of RAW 264.7 • By FACS, 60-70% of the live cells are expressing 24 hrs post-transfection • By Trypan Blue exclusion, the lipofection kills 20-30% of the cells (when the lipid is left on the cells overnight) 5 ug DNA, 10 ul of Lipofectamine2000 in a 6-well plate

  11. Microscopy of YFP Fusion Protein Expression in RAW 264.7 cells EF-1_ YFP-Jnk1 Phase ~35% trnsfxn efficiency EF-1_ YFP-p38MAPK Phase

  12. Western Blot of YFP Fusion Protein Expression in RAW 264.7 YFP control (20ng) EF1_YFP-p38MAPK CMV_YFP-p70S6 EF1_YFP-p70S6 EF1_YFP-Jnk1 CMV_Jnk1-YFP no DNA CMV_YFP EF1_YFP M KDa 120.0 YFP-p70S6kinase 83.8 YFP-Jnk1 YFP-p38MAPK 65.8 50.1 38.6 YFP 27.5 ~ 200,000 cells (or ~20 ug of total protein) loaded per lane

  13. Expression level comparison RAW 264.7, transiently transfected ~100ng of signaling protein per 1x106 WEHI 231, stably infected and selected ~25ng of signaling protein per 1x106

  14. 3T3-L1 Transfection Summary

  15. J774 Transfection Summary

  16. Lipofection of AtT-20 cells 2.5 ug DNA, 10 ul of Lipofectamine2000 (6-well plate) CMV_YFP Phase 70-80% Transfection efficiency

  17. Transfection Summary • Transfection efficiency of RAW 264.7 cells using Lipofectamine2000 is high (~30-70% depending on DNA construct) • Protein expression level is ~4X higher in RAW cells compared WEHI 231 • Transfection efficiency of ~30-70% for 3T3-L1 cells depending on method used • Poor transfection efficiency in J774 cells • High transfection efficiency in AtT-20 cells • All conditions need to be optimized and repeated

  18. Acknowledgements Dallas Cell Lab Robert Hsueh Yan Ni Richard Davis Protein Chemistry Lab Farah El Mazouni Deepa Sethuraman Robert Cox

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