title: Optimization of transfection methods for Huh-7 and Vero cells: comparative study.
abstract: Availability of an efficient transfection protocol is the first determinant in success of gene transferring studies in mammalian cells which is accomplished experimentally for every single cell type. Herein, we provide data of a comparative study on optimization of transfection condition by electroporation and chemical methods for Huh-7 and Vero cells. Different cell confluencies, DNA/reagent ratios and total transfection volumes were optimized for two chemical reagents including jetPEI and Lipofectamine 2000. Besides, the effects of electric field strength and pulse length were investigated to improve electroporation efficiency. Transfection of cells by pEGFP-N1 vector and tracking the expression of GFP by FACS and Fluorescence Microscopy analysis were the employed methods to evaluate transfection efficiencies. Optimized electroporation protocols yielded 63.73 +/- 2.36 and 73.9 +/- 1.6% of transfection in Huh-7 and Vero cells respectively, while maximum achieved level of transfection by jetPEI was respectively 14.2 +/- 0.69 and 28 +/- 1.11% for the same cells. Post transfectional chilling of the cells did not improve electrotransfection efficiency of Huh-7 cells. Compared to chemical based reagents, electroporation showed the superior levels of transfection in both cell lines. The presented protocols should satisfy most of the experimental applications requiring high transfection efficiencies of these two cell lines.
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran.
authors.forename: A
authors.initials: A
authors.lastname: Hashemi
authors.forename: F
authors.initials: F
authors.lastname: Roohvand
authors.forename: M H
authors.initials: MH
authors.lastname: Ghahremani
authors.forename: M R
authors.initials: MR
authors.lastname: Aghasadeghi
authors.forename: R
authors.initials: R
authors.lastname: Vahabpour
authors.forename: F
authors.initials: F
authors.lastname: Motevau
authors.forename: A
authors.initials: A
authors.lastname: Memarnejadian
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D004558:Electric Stimulation; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014709:Vero Cells
chemical_list: D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins