Download - Willie’s Lab Meeting
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#3Willie’s Lab Meeting
Lemmon Lab 2005
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Projects
• High Through-Put Transfections in Neurons
• siRNA
• Plasmids (cDNA)
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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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Transfections
• Lipid Based– Oligofectamine (Lipofectamine 2000 for
siRNA)– Qiagen’s Transmessenger
Lipid MethodLipid Method – – 6565 genes consistently upregulated genes consistently upregulated
ElectroporationElectroporation – – 1111 genes upregulated genes upregulated
• Electroporation– Amaxa– Ambion– BTX
Fedorov Y, King A, Anderson E, Karpilow J, Ilsley D, Marshall W, Khvorova A.
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Electroporations
• First mouse cell electroporation (Neumann) 19__
cosrfEV extm Extent of Permeabilization Pulse AmplitudeDegree of Permeabilization Pulse Duration
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siRNA Concerns
• Interferon Response
• Non Specific Gene Changes
• Cell Death
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(Transfection Solution)
Transfection Plate
The TransfectionsThe Transfections
(96 different targets)
siRNA or Plasmid Library
Robot Re-Array
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Robot Re-Array
(+) Control siRNA (GAPDH Knockdown)(-) Control SiRNA (Non-Silencing)
(48 siRNAs, Transfection Solution)
Transfection Plate
Transfection
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Mouse
Cerebellum
(48 siRNAs, Controls, Transfect Sol.)
Transfection Plate
Cerebellar Granular Neurons(Hydra?)
Transfection
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(Glass Bottom, PreCoated)Assay Plate
(48 siRNAs, Controls, Neurons, Transfect Sol.)
Transfection Plate
ElectroporationElectroporationTransfection
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EXP 1-10Exp Columns 1-10 (80 Samples)
Growth (-) Growth (+)
++++
++++
The AssayThe Assay
NegativeControl
PositiveControl
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Some Early TransfectionsSome Early Transfections
GFP Glia
siRNA Against GFPw/ Jose’s Help
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GFP siRNA
DAPI
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Mean Intensity of GFP to siRNA for each Cell
200
250
300
350
400
0 1000 2000 3000
siRNA
GF P
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eGFP Knockdown ?
GFP Degradation / Hour
0%
20%
40%
60%
80%
100%
0 24 48 72 96 120 144 168 192
Hours
Pe
rce
nt
Re
ma
inin
g
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InnovationsInnovations
Cold Spring Harbor Laboratory
I.N.B.
GAPDH instead of GFP
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GAPDH KnockdownGAPDH Knockdown109_C4_40X siGAPDH, siControl
109_D4_40X siControl Only
Hoechst
Hoechst
siRNA
siRNA
GAPDH
GAPDH
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GAPDH KnockdownGAPDH Knockdown
Exp / Contro l
0.01
0.1
1
10
PBS, HPF INB INB, DH PBS, DH
Av
gIn
t..
[GA
PD
H]
11
1
2
3
4
5
6
7
8
10
9
12
Mo
re K
no
ckd
ow
n
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Hoechst
siRNAJose’s Dark Horse
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New Control Assay (4 Channel)New Control Assay (4 Channel)
Before Fixation
After Fixation
All Nuclei : Hoechst
Dead Nuclei : Sytox Green
siRNA : Alexa Fluor 647
All Nuclei : Hoechst
siRNA : Alexa Fluor 647
Cell : ß-Tubulin Ab, 2° Alexa 488
Knockdown (GAPDH) : Alexa 555
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112.2.Day3112.2.Day3
siControl
All in Solution INB#1
siGAPDH
siControl
siGAPDH
siControl
siControl
siGAPDH
siGAPDH
DH
LDH
x1
x2
LDH
DH
x1
x2
x1
x2
x1
x2
150 150 150 200 200 200 300 300 400 400 500 600
200 400 600200 400 600 150 400 100 250 100 75
siRNA Amount 0.1gμ Additive
Volts
μs
Pulse Conditions: Pulse Voltage, Pulse Length
W.Buchser
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Mid-Recent ResultsMid-Recent ResultsValid Neuron Count
1 2 3 4 5 6 7 8 9 10 11 12
A
B
D
E
F
G
H
C
10
20
30
40
50
60+
0
4
8
12
16
100 200 300 400 50020 100 200 300 400 50020
H6(50)
G6(50)
# o
f C
ells
GAPDH siRNA vs. Control siRNA
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Staining OptimizedStaining Optimized
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Plate / Well IssuesPlate / Well Issues
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Recent ResultsRecent Results
Collaborations / Assistance
• Stephanie - NPCD
• Lawrence Moon (Bunge/Wood)– mRNA
• Darci Moore (Jeff Goldberg)– RGC cDNA
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Staining Issues - TubulinStaining Issues - Tubulin
0
500
1000
1500
2000
2500
0 200 400 600 800 1000
119.1 AD
121.1 AD
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P9 M ouse 24H ours P ost Transfection
Hoechst
eGFP
Merge
B oth from 120.1 .G 9 40X O bjectiveE ffic iency <1%
120.1.G 10.40X
Before Fixation 48Hours