Contents 1 Introduction: 2 Materials: 3 Procedures: 4 Result:

The quality test of the clones of G3&G4

2014/9/20 Test the quality of clones we pick from G3 and G4

Introduction:

In our experiment, not only cell pools but also monoclonal cells are obtained for the further experiments. The monoclonal cells are supposed to have the same insertion sites and copy number which make the system start with fewer variables. What’s more, monoclonal cells with low copy number of EGFP gene is preferred and constant copy number of Cas9 gene. Cells with high copies of EGFP gene may conceal the damage of part of them caused by Cas9 and therefore underestimate the efficiency of Cas9 protein and gRNA. What’s more, how copy number of Cas9 will influence the efficiency of the system is unknown. Different monoclonal cells may give some indications about it. Now, many monoclonal cells from G3 and G4 (both are stably transfected with EGFP gene and Cas9 gene) are obtained. They need to be qualified and some of them will chose for further study.

Materials:

• Monoclonal cells and the pool of G3 (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 (gibco Invitrogen®)
• Endotoxin-free extraced UAS-mCherry-gRNA with gRNA targeting EGFP and 7 UAS.
• DMEM (CORNING) (Hi-clone Thermo) with FBS(gibco Invitrogen) 1µg/ml doxycycline
• DMEM with FBS 1µg/ml and Pen-Strep

Procedures:

1. [2014 Sept. 20th] Observe all monoclonal cells obtained under fluorescence microscope and choose the clones whose fluorescence intensity appears to be lower and passage them on a new plate (generally lower fluorescence intensity indicates lower copy number)
2. [2014 Oct. 3rd] Seed the monoclonal cells on 24 well plate and culture them in 0.5 mL DMEM with 10% FBS, penicillin-Streptomycin and 1000ng/mL doxycycline(must be added to induce the expression of Cas 9)
3. [2014 Oct. 4th] Discard the old medium and add DMEM with 10% FBS and 1000ng/mL doxycycline.
4. Transfect cells with the plasmid 7 UAS encoding gRNA and mCherry using Lipofectamine® 3000 Reagent.
5. Passage cells on the next day, culture cells in DMEM with 10% FBS Penicillin-Streptomycin and 1000ng/mL doxycycline.
6. Observe the cells under a fluorescence microscope and read cells with flow cytometry in the following days.

Result:

The result of flow cytometry.

On the whole, the percentage of the cells with EGFP signal in all clones transfected with plasmid encoding gRNA decline dramatically, decline from 91%- 99% to 50%-79%.

Table 1. The summary of the data of flow cytometry .