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ASSA13-03-29 Optimization of Transfection Conditions For Transfecting pAAV-IRES-hrGFP Plasmid to H9C2 Cell Mediated by Lipofectamine 2000
  1. Liu Xiao-li1,2,
  2. Hong Yuan1,3,
  3. Liu Hai-yan1,2,
  4. Huang Zhi-jun1,3,
  5. Xing Xiao-wei1
  1. 1Clinical Pharmacology Center, The third Xiangya Hospital of Central South University
  2. 2Department of Pharmacy, Central South University
  3. 3Hunan Hypertension Research Center

Abstract

Objective (1) To optimise the conditions of transfecting the liposome-mediated eukaryotic expression plasmid pAAV-IRES-hrGFP to H9C2 cells; (2) To verify the optimised parameters by magnified cell culture and transfecting experiment.

Methods (1) H9c2 cells (spread to 7–9 generation) are cultivated in 96-well plate. Three major factors of 4 levels (decking density (A: 2000/well, 3000/well, 4000/well, 5000/well), the amount of DNA (B: 0.1 ug, 0.2 ug, 0.3 ug, 0.4 ug), and the amount of liposome (C: 0.25 ul, 0.5 ul, 0.75 ul, 1.0 ul) respectively) are considered into the orthogonal test design, which is comprised by 16 groups with additional three control groups (blank control, DNA only, liposome only). After 24h, 48h and 96h of transfection, the efficiencies were examined under a fluorescence microscope. Briefly, under the scope magnified by 1000 times, 10 different fields are randomly selected, in which the number of cells with or without expressing the green fluorescent protein are counted respectively. Then, the transfection efficiency was calculated as the rate:transfection rate = (number of cells expressing green fluorescent protein/the total number of cells in the field of view) × 100%. (2) H9c2 cells (spread to 7–9 generation) are cultured again in 24-well plate, while the total cultivation area was enlarged by 4.00 times (recommended), and 6.67 times separately (according to the area ratio). Then the optimised parameters (4000, 0.1 ug, 0.75 ul) was examined by following transfection.

Results (1) Intuitive analysis showed that the best combination for transfection is A3B2C4, which indicates the parameters will be like this: decking density 4000/well, the amount of DNA 0.2 ug, liposomal dosage 1.0 ul. (2) The transfection efficiency of the 4.00 times group is lower than the condition of the initial 96-well cell culture plate, of which the difference was statistically significant (P < 0.05). At the same time, no significant difference (P > 0.05) between the transfection efficiency of the 6.67 times group and the 96-well cell culture plate conditions is observed.

Conclusions (1) The optimal conditions of transfecting the liposome-mediated eukaryotic expression plasmid pAAV-IRES-hrGFP to H9c2 cells was: decking density 4000/well, the amount of DNA 0.2 ug, the amount of liposomes 1.0 ul. (2) The optimised parameters can be applied to a linear expanded model which is carried out according to the area ratio.

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