Examination of the mCherry expression of the plasmids UAS-mCherry-gRNA

Introduction:

To indicate the plasmid expressing gRNA carries mCherry gene to indicate whether gRNA has entered the cells. Detection of mCherry is important for further experiments. But our fluorescence microscope have no yellow light source which is supposed to excite mCherry. So we firstly try to use green light to excite mCherry. And it is also needed to know when appropriate time for the detection of the signal of mCherry is.

Materials:

• G3 cell (HeLa stably transfected with EGFP gene and Cas9 gene under the control of Tet-on) seeded in 24 well plate, about 90% confluence.
• Lipofectamine® 3000 Reagent
• Opti-MEM
• Endotoxin-free extracted UAS-mCherry-gRNA with (0, 2, 5 or 7) UAS sequence
• DMEM with 10% FBS.
• DMEM with 10% FBS, Penicillin-Streptomycin and 2.8μg/mL blasticindin and 2.0 μg/mL puromycin.

Procedures:

1. [2014 Sept. 14th 22:00]Transfect cells with the four plasmids separately using Lipofectamine® 3000 Reagent.

1. Passage cells on the next day, culture cells in DMEM with 10% FBS Penicillin-Streptomycin.
2. Observe the cells transfected under fluorescence microscope and read the cells with flow cytometry in the following days.

Result:

All of the four UAS plasmids constructed can express mCherry protein normally and the fluorescence of mCherry can be observed. The mCherry signal increased during the first two days, and declined from the third day. After 6 days, nearly no mCherry fluorescence can be detected under the fluorescence microscope. Two or three days after transfection should be the best time to examine the transfection efficiency. It also shows that the lipofectamine 3000 has rather high efficiency of transfection.