Transposon mediated method for stably transfecting non-viral eukaryocyte with high efficiency
A eukaryotic cell and non-viral technology, applied in the genetic field, can solve the problems of high gene cloning technology and time requirements, low stable transfection efficiency, etc., and achieve the effect of simplifying technology and time, and improving efficiency
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Embodiment 1
[0048] Example 1: Transfection and detection of immortalized human primary bronchial epithelial cells (HBEC)
[0049] (1-1) Collect the immortalized HBEC cells grown in several phases, resuspend in 100ul Lonza SF transfection reagent (Lonza), add to the transfection cup, the cell concentration is 10 7 / ml. Add ITR-CMV-GFP vector and EF1-SB100X (2ug+1ug, ratio 2:1, plasmid concentration greater than 0.5ug / ml), or SB-2in1-GFP (3ug, plasmid concentration greater than 0.5ug / ml) flick transfection Mix the staining cup and let it stand for 5 minutes. In the control group, only 2ug ITR-GFP carrier was added.
[0050] (1-2) The CM-130 program of Lonza 4D-Nucleofector (Lonza) was used for electrotransfection of plasmids.
[0051] (1-3) After transfection, add 500ul of medium to the electroporation cup, and use the disposable pipette provided in the kit to add all the cell suspension (about 600ul) into 15ml of complete medium (RPMI1640+10% fetal bovine serum ) in T75 cell culture fla...
Embodiment 2
[0054] Such as Figure 6 Shown is the high-efficiency stable transfection of HBEC cells induced by the SB single-vector system: the proportion of GFP-positive cells detected by flow cytometry after 2 and 10 days of HBEC cell transfection. The results showed that although the efficiencies of transient transfection in the three groups reached about 90% (Day 2), after 10 days (Day 10), the GFP-positive cells in the ITR-CMV-GFP alone transfection group were less than 1%; However, about 20% of the cells were still positive for GFP after transfection with the SB double plasmid. In contrast, after 10 days of expansion of cells transfected with a single plasmid of SB-2in1-GFP, the positive rate of GPF was more than 50%, which proved that the efficiency of stable transfection was significantly improved. Example 2: Transfection and detection of human lung cancer cell line PC-9
[0055] (1-1) Collect PC9 cells grown in several phases, resuspend in 100ul Lonza SE transfection reagent, a...
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