Contents 1 Introduction 2 Materials 3 Procedures: 4 Result:

Test of the first clone of G3 (G3-6)

2014/9/27 Prepare for transfection with A-B toxin!

Introduction

G3 are the cells stably transfected with EGFP gene under the control of EF1a promoter Cas 9 gene under the control of Tet-on system. Now one of the clone have grown and have enough amount of cells for transfection and testing. So 4 plasmids encoding gRNA targeting EGFP will be transfected into G3-6 cells to see the efficiency of Cas9 to cut off the EGFP gene.

Materials

• G3-6: One clone of G3 cell (HeLa stably transfected with EGFP gene and Cas9 gene under the control of Tet-On) seeded in 24-well plate, at about 90% confluence.
• Lipofectamine® 3000 Reagent
• Opti-MEM (gibco Invitrogen®)
• Endotoxin-free extraced UAS-mCherry-gRNA with gRNA targeting EGFP and 0,2,5,7 UAS.
• DMEM (CORNING) (Hi-clone Thermo) with FBS(gibco Invitrogen)
• Complete medium with Penicillin-Streptomycin and 2.8µg/mL blasticindin 2.0µg/mL puromycin and 1µg/mL doxycycline.

Procedures:

[2014 Sept. 14th 11:00pm] Discard the old medium and add 0.5mL DMEM+10%FBS + 1 g/mL doxycycline(for inducing expression of green fluorescence protein) to each well (6 wells in total, four for plasimd transfection, one is added transfection reagent without DNA, one is the control)

1. Transfect cells with those four plasmids with different number of UAS separately using Lipofectamine® 3000 reagent.
2. Passage cells on the next day, culture cells in DMEM+10%FBS+Pen-Strep and 2.8 µg/ml blasticidin 2.0 µg/mL puromycin and 1µg/ul.
3. Observe the cells under a fluorescence microscope and read cells (only the cells transfected with 0UAS and not treated) with a flow cytometer in the following days.

Result:

After transfection, the EGFP fluorescence of some cells became weak or disappeared in the four group of cells transfected with 0UAS, 2 UAS, 5UAS or 7UAS (only the picture of 0UAS is shown, figure 1), which is shown by the result of flow cytometry(the positive percentage decreases from 97.25% to 69.10%) (Figure 2). Unfortunately, puromycin and blasticidin is used to culture the transfected cells.So part of cells loss the EFGP might die, which results in a superficial lower efficiency of EGFP gene cutting than it should be. What is more, the result of this experiment also shows that the plasmid encoding gRNA constructed by our team functions well.