Contents 1 Introduction: 2 Materials: 3 Procedures: 3.1 2014 Sept. 29 3.2 2014 Oct. 2 3.3 2014 Oct. 3 3.4 2014 Oct. 4~7 4 Result 5 Conclusion

Investigation Cas9 protein affect cell growth

2014/9/29 Investigation of the effect of Cas9 protein on the cell growth(The safety of Cas9)!

Introduction:

The Cas9 gene is an exogenous gene for HeLa cells. It is considered that Cas9 protein itself of it combines with small RNAs endogenous in the cells, effecting the cell growth or causing other DNA damages. In this experiment, the cell growth curves of G5 (HeLa stably transfected with Cas9 gene) will be tested and then compared with normal HeLa cells. The double time of the cells which reflects the cell state can also be calculated.

Materials:

• HeLa cells (frozen in liquid nitrogen)
• G5 (HeLa cells stably transfected with Cas9 gene)
• Puromycin (10mg/ml)
• Doxycycline (10mg/ml)
• Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal *bovine serum (FBS) and 1X Penicillin-Streptomycin
• 0.25% trypasin(w/v) with 0.02% EDTA
• 24 well plate

Procedures:

2014 Sept. 29

Thawing and recovering HeLa cells (frozen in liquid nitrogen) and G5 (HeLa cells stably transfected with Cas9 gene), and then culture HeLa cells and G5 in DMEM with 10% FBS and 1X Penicillin-Streptomycin.

2014 Oct. 2

Seed cells in a 24 well plate, 30,000 cells per well.

1. Wash cells with PBS, trypsin digestion, add medium to stop digestion
2. Transfer the cell suspension into 15 mL centrifuge tube and centrifuge at 800rpm for 5 min.
3. Discard the supernatant and add 2 mL complete medium to suspend the cells.
4. Add 1mL PBS to every gap between well
5. 1:10dilution of cells (20 μL cell suspension + 180 μL complete medium)
6. Count the cell concentration with blood counting chamber.
7. Get the cell concentration, take 540,000 G5 cells into a 15 mL centrifuge tube(tube A) and 180,000 HeLa cells into another 15 mL centrifuge tube(tube B).
8. Add complete medium to tube A to get a total volume of 9mL and tube B to get a total volume of 3mL.
9. Mix well the cell suspension by shaking and pipette gently.
10. Add 0.5 mL of cell suspension to each well. (The first row is HeLa cells and the next three rows are G5).
11. Observe the cells on the next day, and the cells grow well and uniformly.

2014 Oct. 3

Discard the old medium and add new medium to the cells (0.5 mL per well).

1. Add complete medium with 1μg/mL doxycycline to the first row, HeLa cells and the second row, G5 cells.
2. Add complete medium with 10μg/mL puromycin to the third row, G5 cells.
3. Add complete mediume with 1μg/mL doxycycline and 10μg/mL puromycin to the forth row, G5 cells.

2014 Oct. 4~7

In the following days, replace the medium every two or three days, and count the cells every day (for each group, one well per day)as the procedures stated following: discard the medium, wash with PBS twice, digest cells with 100μL trypsin at 37℃ for 2 min, add complete medium of known volume to the cells, pipette cell solution gently for at less 20 times to suspend the cells thoroughly and then transfer the cells suspension into a 1.5mL centrifuge tube, calculate the cell concentration with blood counting chamber or countstar, and calculate the cell number by multiply the cell concentration and the total volume of trypsin and medium.

Result

For HeLa + Dox, y = 51550e^(0.0264X)(y is the cell number and x is time with the unit hour), double time: 26.25 hours;
For G5 + Dox, y = 52419e^(0.022X) , double time: 31.55 hours;
For G5 + puro, y = 52257e^(0.0241X) , double time: 28.74 hours;
For G5 + Dox + puro, y = 51763e^(0.0221X) , double time: 31.35hours.
Then the double time can be obtained .
For G5+ Dox and G5 + Dox + Puro, the matched cell growth curves are very similar. It indicates that the existence of puromycin does not affect the growth of G5.
The coefficient of x indicates the rate of cell growth. For G5, the coefficient ranges from 0,022 to 0.0241, while that of HeLa is 0.0264. It shows that normal HeLa cells grow faster than G5 cells. But it might also be an accidental event. It is also shown that the cells with Cas 9 expression(G5 + Dox, and G5 + Dox + puro) grows a little slower. But no obvious cell death has been detected.

Conclusion

1. G5 cells which has been stably transfected with Cas9 gene grows a little slower than normal HeLa cells (double time 31.55 hours vs. 26.25 hours). The gene transfection may affect the cells to some extent, which could be result from the treatment of lipofectamine 3000 or the exogenous gene insertion affects part of cells in G5.
2. The expression of Cas9 seems to make G5 grows a little slower (31.55 hours and 31.55 hours vs. 28.74 hours) but no other serious influence has been observed. One reasonable explanation is that the expression of Cas 9 protein increase the burden of cell metabolism, and therefore slower the progress of the cell cycle. So this experiment indicates that the transfection and the expression of Cas9 gene are '''safe''' to HeLa cells basically.