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