Volume 1, Issue 2 ((Journal of Clinical and Basic Research (JCBR) 2017)                   jcbr 2017, 1(2): 16-20 | Back to browse issues page

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Abstract:   (4215 Views)

Introduction: There are several methods for delivery of vectors into eukaryotic cell lines. Transfection with liposomes is an easy and accessible way. Lipofectamine 2000 is a transfection reagent with liposome structure. Despite having a specific protocol, the volume of this reagent should be optimized for use in different cell lines. The aim of this study was to optimize transfection of KYSE-30 cell line with pEGFP-NI vector using Lipofectamine 2000. Materials and Methods: The vector was purified by plasmid extraction kit. Transfection of the KYSE-30 cell line was done using Lipofectamine 2000 and different concentrations of the vector. Expression of green fluorescent proteins (GFP) was evaluated by fluorescence microscopy, and later analyzed with ImageJ software. Results: Optimized concentration of plasmid (5μg) and volume of Lipofectamine 2000 (6μl) were determined for KYSE-30 cell line. GFP plasmid transfection using the determined values showed more than 65% efficiency in the KYSE-30 cell line. The quantity of DNA per transfection and volume of reagent were identified as essential factors for a successful transfection. Conclusions: This study shows that lipofection with lipofectamine 2000 is an efficient method of gene delivery into KYSE-30 cell line.

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References
1. Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. 2010;397(8):3173-8. [DOI:10.1007/s00216-010-3821-6]
2. Dalby B, Cates S, Harris A, Ohki EC, Tilkins ML, Price PJ, et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods. 2004;33(2):95-103. [DOI:10.1016/j.ymeth.2003.11.023]
3. Oudrhiri N, Vigneron J-P, Peuchmaur M, Leclerc T, Lehn J-M, Lehn P. Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo. Proceedings of the National Academy of Sciences. 1997;94(5):1651-6. [DOI:10.1073/pnas.94.5.1651]
4. Labat-Moleur F, Steffan A-M, Brisson C, Perron H, Feugeas O, Furstenberger Pa, et al. An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene therapy. 1996;3(11):1010-7.
5. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. Journal of Biological Chemistry. 1995;270(32):18997-9007. [DOI:10.1074/jbc.270.32.18997]
6. Zuhorn IS, Kalicharan R, Hoekstra D. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. Journal of Biological Chemistry. 2002;277(20):18021-8. [DOI:10.1074/jbc.M111257200]
7. Yamano S, Dai J, Moursi AM. Comparison of Transfection Efficiency of Nonviral Gene Transfer Reagents. Molecular Biotechnology. 2010;46(3):287-300. [DOI:10.1007/s12033-010-9302-5]
8. Abràmoff MD, Magalhães PJ, Ram SJ. Image processing with ImageJ. Biophotonics international. 2004;11(7):36-42.
9. Cui S, Zhang S, Chen H, Wang B, Zhao Y, Zhi D. The Mechanism of Lipofectamine 2000 Mediated Transmembrane Gene Delivery. Engineering. 2012; 5: 172-175 [DOI:10.4236/eng.2012.410B045]
10. Avci-Adali M, Behring A, Keller T, Krajewski S, Schlensak C, Wendel HP. Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression. Journal of biological engineering. 2014;8(1):1. [DOI:10.1186/1754-1611-8-8]
11. Boot HJ, Dokic K, Peeters BPH. Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus. Journal of Virological Methods. 2001;97(1–2):67-76. [DOI:10.1016/S0166-0934(01)00340-8]
12. Maurisse R, De Semir D, Emamekhoo H, Bedayat B, Abdolmohammadi A, Parsi H, et al. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC biotechnology. 2010;10(1):9. [DOI:10.1186/1472-6750-10-9]
13. Narayanan K, Lee CW, Radu A, Sim EUH. Escherichia coli bactofection using Lipofectamine. Analytical biochemistry. 2013;439(2):142-4. [DOI:10.1016/j.ab.2013.04.010]
14. Ward CM, Stern PL. The Human Cytomegalovirus Immediate‐Early Promoter is Transcriptionally Active in Undifferentiated Mouse Embryonic Stem Cells. Stem cells. 2002;20(5):472-5. [DOI:10.1634/stemcells.20-5-472]
15. Shabani M, Hemmati S, Hadavi R, Amirghofran Z, Jeddi-Tehrani M, Rabbani H, et al. Optimization of gene transfection in murine myeloma cell lines using different transfection reagents. Avicenna journal of Medical Biotechnology. 2010;2(3):123-30.
16. Salimzadeh L, Jaberipour M, Hosseini A, Ghaderi A. Non-viral transfection methods optimized for gene delivery to a lung cancer cell line. Avicenna Journal of Medical Biotechnology. 2013;5(2):68.
17. Hashemi A, Roohvand F, Ghahremani M, Aghasadeghi M, Vahabpour R, Motevali F, et al. Optimization of transfection methods for Huh-7 and Vero cells: A comparative study. Cytology and Genetics. 2012;46(6):347-53. [DOI:10.3103/S0095452712060035]

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