To Be Planned:
Lyn transfection:
Do we need to transfect any additional Lyn or can we rely on endogenous levels of Lyn? If we cannot rely on endogenous Lyn, what concentrations on Lyn are sufficient for functioning of BCR.
Transfection protocols:
Characterizing effectiveness of different transfection protocols. We want to test Lipofectamine transfection with adhered cells vs. cells in suspension and possibly with the new transfection reagent jetprime.
Amounts of transfected plasmids:
What are the minimum amounts of plasmid we need in a multiple plasmid transfection? hEF1a:rTta, TRE:gal4vp16, gal4UAS:X1FP, hEF1a:X2FP, hEF1a:X3FP. Possible concentrations: 10ng, 25ng, 50ng, 100ng, 250ng, 500ng, 1000ng.
Expression level optimization:
LacI controlled BCR expression and TRE controlled Syk-TEVp expression level optimization.
+ve control: hEF1a:Gal4VP16 --> XFP.