Establishment and verification of transformation conditions for mouse RAW264. 7 macrophages by electroporation
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1.Department of Sports and Health Sciences, Nanjing Sport Institute, Nanjing 210014, China. 2. Department of Exercise Physiology, Beijing Sport University, Beijing 100084. 3. Jiangsu Research Institute of Sports Science, Nanjing 210033

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    Abstract:

    Objective To screen and validate the conditions for transforming Tribbles Pseudokinase 3 (TRIB3) overexpression plasmids and siRNA into RAW264. 7 cells by electroporation, to establish the best electroporation scheme for RAW264. 7 cells. Methods By changing the conditions of pulse voltage, pulse duration and plasmid concentration,TRIB3 overexpression plasmid and TRIB3 siRNA were transformed into RAW264. 7 cells. Cell morphology was observed by phase contrast microscopy. Cell viability was detected by CCK-8, and TRIB3 protein expression was detected by Western blot. Results (1) When transformed with different pulse voltages, there was no difference in cell morphology and survival rate between 110 and 120 mV groups and the control group. The cell survival rate in the 130 and 140 mV groups were significantly lower than in the control group (P< 0. 01). TRIB3 protein expressions in the 120, 130 and 140 mV groups were significantly increased than that in the control group (P < 0. 05). ( 2) When transformed with different pulse durations, there was no difference in cell morphology and survival rate between the 10 and 20 ms groups and the control group. The cell survival rate in the 30 ms group was significantly lower than that in the control group (P< 0. 01). The expressions of TRIB3 in the 20 and 30 ms groups were significantly increased than that in the control group (P< 0. 01). (3) When transformed with different plasmid concentrations, the cell morphology and survival rate of the 2 and 4 μg groups were not different from the control group. The cell survival rate of the 6 μg group was lower than that in the control group (P< 0. 05), and the expression of TRIB3 in the 4 μg group was significantly increased than that in the control group (P< 0. 01). (4) When transformed with TRIB3 siRNA at different concentrations, the expression of TRIB3 in the 10 nmol / L group was only 0. 19 times that in the control group ( P < 0. 01). Conclusions In RAW264. 7 cells, the TRIB3 overexpression plasmid ( 4 μg / sample) and TRIB3 siRNA ( 10 nmol / L) were transfected by electroporation under the conditions of 120 mV and 20 ms, which ensured a high cell survival rate and significantly changed the protein expression level of the target gene.

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History
  • Received:September 09,2019
  • Revised:
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  • Online: April 29,2020
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