Part:BBa_K2597001
TsgRNA-dCas9-VP64
BBa_K259701 consists of TsgRNA designed to target telomeric repeat sequence and artificial transcription factor dCas9-VP64 to activate transcription of effector gene on the vector. We cloned the linear TsgRNA fragments target telomere into plasmid pcDNA-dCas9-VP64 to generate plasmid TsgRNA-dCas9-VP64 for convenience of using and improvement in transfection efficiency. When transfected in cells with high telomerase activity, dCas9-VP64 could bind the telomeric repeat sequence synthesized by telomerase thus activate the expression of effector gene Cas9, leading to its cutting on telomeres and the apoptosis of cancer cells.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experiment design
For characterization, TsgRNA-dCas9-VP64 was transfected in various cell lines as part of the Tage system, testing its function in activating the expression of effector genes . We verify the function of the system in killing cancer cells by utilizing Cas9 as the effector gene to cut telomeres. We transfected cells with TsgRNA-Cas9-VP64, HO-site TMCP and C1-HO. The cells were stained after transfection andcultivation with Acridine orange and then observed and photographed with fluorescent microscope. The photographs could show us the change in cell density, thus showing whether the system is effective in killing cancer cells without harming normal cells.
The results reveal that the Tage system can induce the significant death of transfected cancer cells HepG2, as it shows obvious decrease in density, while exerts no effects on normal cells HL7702. As controls, the cells are also transfected with lipofectin, and the results confirms the function of TsgRNA-dCas9-VP64 in expressing the effector gene Cas9 as a necessary part of Tage system.
Methods
HepG2 was cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal calf serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. HL7702 was cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium, supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin.
DNA was aliquoted into individual tubes before transfection. For each transfection, cells were cultured with 500 L of Opti-MEM at 37 °C for 30 min when grown to a density of 2×105 cells/well. A stock solution of 50 L of Opti-MEM, 500 ng of total DNA and 50 L of Opti-MEM, 2 L of Lipofectamine 2000per transfection was made respectively. The solution was then vortexed respectively and incubated for 5 minutes at room temperature. Thereafter, the Opti-MEM/Lipofectamine solution was added to the individual aliquots of DNA stocked in 50 L of Opti-MEM, vortexed, and incubated for 20 minutes at room temperature before being added to each well. After incubated for 5 h, the medium of each well was replaced with 500 L of fresh DMEM or RPMI 1640 medium containing 10% FBS.
After transfection, the cells were further incubated at 37 °C and 5% CO2 for another 24 h. Cells were stained with Acridine orange. Cells were washed twice with PBS, then stained with 100 g/mL acridine orange for 10 min at room temperature. Cells were then observed and photographed with a fluorescent microscope at the constant magnification of 200×.
| None |