willie’s lab meeting

#3Willie’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

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.

Electroporations

• First mouse cell electroporation (Neumann) 19__

cosrfEV extm Extent of Permeabilization Pulse AmplitudeDegree of Permeabilization Pulse Duration

siRNA Concerns

• Interferon Response

• Non Specific Gene Changes

• Cell Death

(Transfection Solution)

Transfection Plate

The TransfectionsThe Transfections

(96 different targets)

siRNA or Plasmid Library

Robot Re-Array

Robot Re-Array

(+) Control siRNA (GAPDH Knockdown)(-) Control SiRNA (Non-Silencing)

(48 siRNAs, Transfection Solution)

Transfection Plate

Transfection

Mouse

Cerebellum

(48 siRNAs, Controls, Transfect Sol.)

Transfection Plate

Cerebellar Granular Neurons(Hydra?)

Transfection

(Glass Bottom, PreCoated)Assay Plate

(48 siRNAs, Controls, Neurons, Transfect Sol.)

Transfection Plate

ElectroporationElectroporationTransfection

EXP 1-10Exp Columns 1-10 (80 Samples)

Growth (-) Growth (+)

++++

++++

The AssayThe Assay

NegativeControl

PositiveControl

Some Early TransfectionsSome Early Transfections

GFP Glia

siRNA Against GFPw/ Jose’s Help

GFP siRNA

DAPI

Mean Intensity of GFP to siRNA for each Cell

200

250

300

350

400

0 1000 2000 3000

siRNA

GF P

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

InnovationsInnovations

Cold Spring Harbor Laboratory

I.N.B.

GAPDH instead of GFP

GAPDH KnockdownGAPDH Knockdown109_C4_40X siGAPDH, siControl

109_D4_40X siControl Only

Hoechst

Hoechst

siRNA

siRNA

GAPDH

GAPDH

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

Hoechst

siRNAJose’s Dark Horse

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

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

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

Staining OptimizedStaining Optimized

Plate / Well IssuesPlate / Well Issues

Recent ResultsRecent Results

Collaborations / Assistance

• Stephanie - NPCD

• Lawrence Moon (Bunge/Wood)– mRNA

• Darci Moore (Jeff Goldberg)– RGC cDNA

Staining Issues - TubulinStaining Issues - Tubulin

0

500

1000

1500

2000

2500

0 200 400 600 800 1000

119.1 AD

121.1 AD

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