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Willie’s Lab Meeting

#3. Willie’s Lab Meeting. Lemmon Lab 2005. Projects. High Through-Put Transfections in Neurons siRNA Plasmids (cDNA). HTP Neuron Transfections. Why? Develop methods to screen target genes for any assay Combinatorial strategies require many iterations to find optimal ratios.

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Willie’s Lab Meeting

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  1. #3 Willie’s Lab Meeting Lemmon Lab 2005

  2. Projects • High Through-Put Transfections in Neurons • siRNA • Plasmids (cDNA)

  3. HTP Neuron Transfections • Why? • Develop methods to screen target genes for any assay • Combinatorial strategies require many iterations to find optimal ratios

  4. Transfections • Lipid Based • Oligofectamine (Lipofectamine 2000 for siRNA) • Qiagen’s Transmessenger • Electroporation • Amaxa • Ambion • BTX Lipid Method – 65 genes consistently upregulated Electroporation – 11 genes upregulated Fedorov Y, King A, Anderson E, Karpilow J, Ilsley D, Marshall W, Khvorova A.

  5. Electroporations • First mouse cell electroporation (Neumann) 19__ Extent of Permeabilization  Pulse Amplitude Degree of Permeabilization  Pulse Duration

  6. siRNA Concerns • Interferon Response • Non Specific Gene Changes • Cell Death

  7. Transfection Plate (Transfection Solution) The Transfections siRNA or Plasmid Library (96 different targets) Robot Re-Array

  8. Transfection (+) Control siRNA (GAPDH Knockdown) (-) Control SiRNA (Non-Silencing) Robot Re-Array Transfection Plate (48 siRNAs, Transfection Solution)

  9. Transfection Cerebellum Cerebellar Granular Neurons (Hydra?) Mouse (48 siRNAs, Controls, Transfect Sol.) Transfection Plate

  10. Transfection Electroporation Transfection Plate (48 siRNAs, Controls, Neurons, Transfect Sol.) Assay Plate (Glass Bottom, PreCoated)

  11. The Assay + + + + + + + + Growth (+) Growth (-) Negative Control Positive Control EXP 1-10 Exp Columns 1-10 (80 Samples)

  12. Some Early Transfections GFP Glia siRNA Against GFP w/ Jose’s Help

  13. 400 Mean Intensity of GFP to siRNA for each Cell 350 P F 300 G 250 200 0 1000 2000 3000 siRNA

  14. eGFP Knockdown ?

  15. Innovations Cold Spring Harbor Laboratory I.N.B. GAPDH instead of GFP

  16. Hoechst siRNA GAPDH Hoechst siRNA GAPDH GAPDH Knockdown 109_C4_40X siGAPDH, siControl 109_D4_40X siControl Only

  17. GAPDH Knockdown More Knockdown

  18. Jose’s Dark Horse siRNA Hoechst

  19. New Control Assay (4 Channel) Before Fixation Dead Nuclei : Sytox Green All Nuclei : Hoechst siRNA : Alexa Fluor 647 After Fixation All Nuclei : Hoechst siRNA : Alexa Fluor 647 Cell : ß-Tubulin Ab, 2° Alexa 488 Knockdown (GAPDH) : Alexa 555

  20. Yan Shi

  21. 112.2.Day3 siRNA Amount 0.1g μ Additive siGAPDH x1 siControl x1 siGAPDH x2 siControl x2 siControl x1 DH siGAPDH x1 DH siControl x2 LDH siGAPDH x2 LDH Volts 150 150 150 200 200 200 300 300 400 400 500 600 μ s 200 400 600 200 400 600 150 400 100 250 100 75 Pulse Conditions: Pulse Voltage, Pulse Length All in Solution INB#1 W.Buchser

  22. A 60+ B 50 C 40 D 30 E F 20 16 G 10 H 12 s l l H6(50) 1 2 3 4 5 6 7 8 9 10 11 12 e C 8 G6(50) f o # 4 0 20 20 100 100 200 200 300 300 400 400 500 500 Mid-Recent Results Valid Neuron Count GAPDH siRNA vs. Control siRNA

  23. Staining Optimized

  24. Plate / Well Issues

  25. Recent Results Collaborations / Assistance • Stephanie - NPCD • Lawrence Moon (Bunge/Wood) • mRNA • Darci Moore (Jeff Goldberg) • RGC cDNA

  26. 2500 2000 1500 1000 500 0 0 200 400 600 800 1000 Staining Issues - Tubulin 119.1 AD 121.1 AD

  27. Before Fixation 48Hours

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