Part:BBa_K2597002
Homothallic switching endonuclease(C1-HO)
BBa_K259702 is constructed to express HO enzyme that could cut a blunt-ended effector containing HO recognizable site (HOsite-TMCP) to produce a stick end of 3’-TTGT-5’. The stick end could be recognized by telomerase, leading to the double-stranded telomeric repeat sequence synthesis.
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, we construct the vector that could express HO protein to test if it could cut a blunt-ended effector containing a HO site to produce a stick end of 3’-TTGT-5’. We transfected the cells with Tage system consisting of four vector including C1-HO, HOsite-bTMEP, TsgRNA and dCas9-VP64. The cells were cultivated after transfection and then observed and photographed with a fluorescence microscope. The fluorescence intensity of cells was quantified with a flow cytometry and the mean fluorescent intensity was calculated by BD software.
The results reveal that the ZsGreen protein was expressed by telomerase positive cells HepG2, but not by the telomerase negative cells HL7702, verifying the process of producing stick end and C1-HO’s effectiveness.
In further experiments, C1-HO was transfected into cells with TsgRNA, dCas9-VP64 and HOsite-TMCP, testing Tage system’s function in killing cancer cells. The cells were stained after transfection and cultivation with Acridine orange and then observed and photographed with fluorescent microscope.
The results reveal that the cancer cell HepG2 can be significantly killed by the Tage system; however, the growth of normal cells HL7702 was not affected by the Tage system, indicating the function of C1-HO as expressed HO protein can cut the T-HOsite- TMCP plasmid to produce a telomerase recognizable stick end. (Figure 2)
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. In the first experiment, Cells were observed and photographed with a fluorescence microscope at a constant magnification of 200×. The fluorescence intensity of cells was quantified with a flow cytometry and the mean fluorescent intensity (MFI) was calculated by BD software of flow cytometry.
In the second experiment, 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×.
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