Optimization of Triple Plasmids Transfection into HEK293 Cells Mediated by Polyethylenimine_Journal of Biomedical Engineering

Authors：

FUQiang ¹ , LIYan ² ,  ZHENGZhaofen ¹ , LIUAizhong ² , YUANZhenhua ³ , PENGJianqiang ¹ , HEJin ¹

1. Department of Cardiology, Hunan Provincial People's Hospital, Hunan Normal University, Changsha 410005, China;
2. Department of Epidemiology and Health Statistics, School of Public Health, Central South University, Changsha 410078, China;
3. Institute of Biomedicine and Biotechnology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China;

Corresponding author：

ZHENGZhaofen, Email: xyxnzzf@126.com

Keywords：

polyethyleneimine; adeno-associated virus; HEK293 cell; transfection efficiency; optimization

DOI：

10.7507/1001-5515.20150025

Video：

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In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6.0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4μg/well with PEI/plasmid ratio of 3:1~5:1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.

Citation： FUQiang, LIYan, ZHENGZhaofen, LIUAizhong, YUANZhenhua, PENGJianqiang, HEJin. Optimization of Triple Plasmids Transfection into HEK293 Cells Mediated by Polyethylenimine. Journal of Biomedical Engineering, 2015, 32(1): 137-141. doi: 10.7507/1001-5515.20150025 Copy

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7.	HILDINGER M, BALDI L, STETTLER M, et al. High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells[J]. Biotechnol Lett, 2007, 29(11): 1713-1721.
8.	FENG L, GUO M, ZHANG S, et al. Improvement in the suspension-culture production of recombinant adeno-associated virus-LacZ in HEK-293 cells using polyethyleneimine-DNA complexes in combination with hypothermic treatment[J]. Biotechnol Appl Biochem, 2008, 50(Pt 2): 121-132.
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13.	HUNTER A C. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity[J]. Adv Drug Deliv Rev, 2006, 58(14): 1523-1531.
14.	MOGHIMI S M, SYMONDS P, MURRAY J C, et al. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy[J]. Mol Ther, 2005, 11(6): 990-995.

1. GRIEGER J C, SAMULSKI R J. Adeno-associated virus vectorology, manufacturing, and clinical applications[J]. Methods Enzymol, 2012, 507: 229-254.
2. MUELLER C, FLOTTE T R. Clinical gene therapy using recombinant adeno-associated virus vectors[J]. Gene Ther, 2008, 15(11): 858-863.
3. MINGOZZI F, HIGH K A. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges[J]. Nat Rev Genet, 2011, 12(5): 341-355.
4. XIAO X, LI J, SAMULSKI R J. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus[J]. J Virol, 1998, 72(3): 2224-2232.
5. DALBY B, CATES S, HARRIS A, et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications[J]. Methods, 2004, 33(2): 95-103.
6. WALTERS R W, DUAN D, ENGELHARDT J F, et al. Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo[J]. J Virol, 2000, 74(1): 535-540.
7. HILDINGER M, BALDI L, STETTLER M, et al. High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells[J]. Biotechnol Lett, 2007, 29(11): 1713-1721.
8. FENG L, GUO M, ZHANG S, et al. Improvement in the suspension-culture production of recombinant adeno-associated virus-LacZ in HEK-293 cells using polyethyleneimine-DNA complexes in combination with hypothermic treatment[J]. Biotechnol Appl Biochem, 2008, 50(Pt 2): 121-132.
9. GRIMM D, KERN A, RITTNER K, et al. Novel tools for production and purification of recombinant adenoassociated virus vectors[J]. Hum Gene Ther, 1998, 9(18): 2745-2760.
10. RUDOLPH C, LAUSIER J, NAUNDORF S, et al. In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers[J]. J Gene Med, 2000, 2(4): 269-278.
11. AKINC A, THOMAS M, KLIBANOV A M, et al. Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis[J]. J Gene Med, 2005, 7(5): 657-663.
12. ZABNER J, FASBENDER AJ, MONINGER T, et al. Cellular and molecular barriers to gene transfer by a cationic lipid[J]. J Biol Chem, 1995, 270(32): 18997-19007.
13. HUNTER A C. Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity[J]. Adv Drug Deliv Rev, 2006, 58(14): 1523-1531.
14. MOGHIMI S M, SYMONDS P, MURRAY J C, et al. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy[J]. Mol Ther, 2005, 11(6): 990-995.

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Optimization of Triple Plasmids Transfection into HEK293 Cells Mediated by Polyethylenimine

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