Transfection of alternative cell lines

RAW 264.73T3-L1J774

AtT-20

Expression of YFP in Electroporated WEHI 231 cells

30-60% transfection efficiency

Phase CMV_YFP

Expression of YFP in electroporated WEHI 231 and HEK293 cells

M

YFP positive control Ctrl YFP

1 YFP2

Ctrl YFP1 YFP2

WEHI 231 293

63.8

49.5

37.4

26.0

20.5

KDa

YFP

Total cell lysate from ~1 mil cells per lane

Anti-GFP (JL-8) blot

Expression of YFP-Akt in Electroporated WEHI 231 cells

Phase EF1_YFP-Akt

~30% transfection efficiency

M

YFP positive control Ctrl YFP-Akt

WEHI-231

63.8 49.5

37.4

26.0

20.5

KDa

YFP-Akt

293

Ctrl YFP-Akt

YFP

80.9113.7182.9

Expression of YFP-Akt in electroporated WEHI 231 and HEK293 cells

Anti-GFP (JL-8) blot

Total cell lysate from ~1 mil cells per lane

Expression of FLAG-Stat3 in retrovirus infected and selected WEHI 231

~200,000 cells in TCL lane ~24 mil cells in IP lane

Anti-Flag Blot

83.8

120.0

M

65.8

TCL IP

FLAG-Stat3

Transfection Tests on Alternative Cells

• Lipofectamine2000• FuGene6• Effectene• SuperFect• GenePorter2

• Electroporation• Retrovirus infection

• CMV_YFP• EF1_YFP

Method

% of Live Cells

Expressing by FACS

LF2000 50-70%

Electroporation

20%

FuGene6 6%

Effectene 3%

GenePorter 2%

SuperFect 2%

RAW264.7 Transfection Summary

Lipofection of RAW 264.7

Phase CMV_YFP

5 ug DNA, 10 ul of Lipofectamine2000 (6-well plate)

Phase EF-1_YFP

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 Lipofectamine2000in a 6-well plate

Microscopy of YFP Fusion Protein Expression in RAW 264.7 cells

Phase EF-1_YFP-Jnk1

Phase EF-1_YFP-p38MAPK

~35% trnsfxn

efficiency

Western Blot of YFP Fusion Protein Expression in RAW 264.7

KDaM

83.8

50.1

120.0

38.6

27.5YFP

YFP

cont

rol

(20ng

)no

DN

A

EF1_Y

FP-Jnk

1C

MV_J

nk1-Y

FPEF

1_Y

FP-p

70S6

CM

V_Y

FP-p

70S6

EF1_Y

FP-

p38M

APK

CM

V_Y

FPEF

1_Y

FP

YFP-p70S6 kinase YFP-Jnk1 YFP-p38MAPK 65.8

~ 200,000 cells (or ~20 ug of total protein) loaded per lane

RAW 264.7, transiently transfected

~100ng of signaling protein per 1x106

WEHI 231, stably infected and selected

~25ng of signaling protein per 1x106

Expression level comparison

3T3-L1 cells

Retrovirus 69%

LF2000 36%

Effectene 20 %

FuGene6 17 %

3T3-L1 Transfection Summary

J774 cells

Retrovirus 8%

FuGene6 5%

GenePorter 4%

Electroporation

2%

LF2000 1%

SuperFect 1%

Effectene 0%

J774 Transfection Summary

Lipofection of AtT-20 cells

Phase CMV_YFP

2.5 ug DNA, 10 ul of Lipofectamine2000 (6-well plate)

70-80% Transfection efficiency

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

Acknowledgements

Dallas Cell LabRobert Hsueh

Yan NiRichard Davis

Protein Chemistry LabFarah El Mazouni

Deepa SethuramanRobert Cox