Part:BBa_K2597000
HOsite-TMCP (HO recognizable site, Cas9)
BBa_K2597000 consists of Homothallic switching endonuclease (HO) recognizable site, Mini-TK promoter, Cas9 and poly A, as we named to be HOsite-TMCP. This part codes for Cas9 gene and expresses Cas9 protein, aiming to cut the telomeres of cancer cells under the guidance of telomerase-targeting sgRNA. HO recognizable site could be recognized by HO enzyme to produce the four-base overhang (3’-TTGT-5’), which would be recognized by telomerase and lead to the synthesis of double-stranded telomeric repeat sequence.
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, HOsite-TMCP was transfected into various cancer cell lines and normal cell lines as part of Tage system to test its function in expressing Cas9 protein and cute the telomeres of cancer cells.
We transfect HOsite-TMCP together with TsgRNA-dCas9-VP64 and C1-HO into different cell lines to test the Tage‘s function in selectively 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 cell HL7702 was not affected by the Tage system. As controls, the cells are also transfected with lipofectin, and the result confirms the function of HOsite-TMCP in expressing Cas9 as a necessary part of Tage system. (Figure 1)
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×.
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